bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of Utility of the Lysis-Centrifugation Blood Culture Method for Detection of Fungemia in Immunocompromised Cancer Patients
Authors: Creger Richard J. , Weeman Kisa E. , Jacobs Michael R. , Morrissey Anne , Parker Pamela , Fox Robert M. , Lazarus Hillard M.
Published: December 2019

We retrospectively compared the utility of a fungal isolation device (Isolator) versus conventional techniques for recovering fungal organisms from blood cultures obtained from neutropenic cancer patients. Positive cultures were deemed true pathogens, possible pathogens, or contaminants according to laboratory and clinical criteria. Fifty-three patients had 66 positive blood cultures for fungi, nine on multiple occasions. In 20 episodes true pathogens were recovered, 6 from broth medium alone, 4 from the Isolator system alone, and 10 from both systems. False-negative cultures were noted in 4 of 20 (20%) cases in which broth medium was used and in 6 of 20 (30%) cases in which the Isolator system was used. Possible pathogens were detected in 4 of 66 blood culture-positive cases. Forty-two positive cultures were considered contaminants, 1 collected from standard medium and 41 of 42 (98%) which grew only in Isolators. Eleven of 18 patients with true fungal infections expired as a result of infection, while 4 of 33 patients with a contaminant expired, none from a fungal cause. We do not advocate the routine use of Isolator tubes in the evaluation of the febrile, neutropenic patient due to the high rates of false positives and of contamination.
Detection of Measles Virus mRNA from Autopsied Human Tissues
Authors: Katayama Yuko , Kohso Kiyoyasu , Nishimura Akiyoshi , Tatsuno Yoshitsugu , Homma Morio , Hotta Hak
Published: December 2019

By reverse transcription-PCR, measles virus (MV) mRNA was detected in the brain, kidney, spleen, liver, and lung tissues obtained from 23 (45.1%) of 51 autopsy subjects, with the detection rates of each tissue ranging from 8 to 20%. Sequence analysis revealed frequent mutations in the corresponding viral protein. These results suggest that MV mutants commonly persist in apparently healthy individuals.
Identification of
Encephalitozoon intestinalis
in Travelers with Chronic Diarrhea by Specific PCR Amplification
Authors: Raynaud Laurent , Delbac Frederic , Broussolle Veronique , Rabodonirina Meja , Girault Veronique , Wallon Martine , Cozon Gregoire , Vivares Christian P. , Peyron François
Published: December 2019

With the use of Weber’s modified trichrome and Uvitex 2B techniques, spores of microsporidia were detected in the stools of four travelers presenting clinically with chronic diarrhea. The general health of these patients was not impaired, and human immunodeficiency virus screening was negative. Immune evaluation, including the study of lymphocytic subpopulations, assay of serum immunoglobulins, and an intradermal multitest, showed normal results. Molecular identification of microsporidian species was based on the PCR amplification of a small-subunit rRNA sequence followed by
Hin
fI endonuclease restriction.
Encephalitozoon intestinalis
microsporidiosis was thus shown in two of the four patients examined. In two patients, therapy based on albendazole made stools devoid of microsporidian spores without influence on the intestinal disorders. The pathogenic role of
E. intestinalis
in immunocompetent individuals remains to be demonstrated.
Characterization of Gentamicin-Susceptible Strains of Methicillin-Resistant
Staphylococcus aureus
Involved in Nosocomial Spread
Authors: Lemaître Nadine , Sougakoff Wladimir , Masmoudi Afef , Fievet Marie-Hélène , Bismuth Roland , Jarlier Vincent
Published: December 2019

We report an outbreak of epidemic
Staphylococcus aureus
strains characterized by an unusual heterogeneous resistance to methicillin and resistance to tobramycin but susceptibility to gentamicin (gentamicin-susceptible methicillin-resistant
S. aureus
[GS-MRSA]), contrasting with gentamicin-resistant homogeneous MRSA (GR-MRSA) that have been endemic in our hospital since the 1970s. A total of 97 GS-MRSA strains, which were shown by DNA hybridization to carry the
mecA
and
ant(4′)-Ia
genes, were studied. The 40 GS-MRSA strains isolated at the beginning of the outbreak (January 1992 to June 1993) were typed by using resistance patterns, phage typing, serotyping, and pulsed-field gel electrophoresis and were compared with GR-MRSA and methicillin-susceptible
S. aureus
(MSSA) strains isolated during the same period. Two dominant clones, A::1 and B::3, and one minor clone, C::5, were identified among the 40 GS-MRSA strains, according to pulsotypes (A to C) and their resistance patterns (1, 3, and 5), which were distinguishable from those of GR-MRSA and MSSA strains. A selection of 57 GS-MRSA strains, isolated from 1994 to 1996, were clustered in the same three clones. However, their distribution had changed in comparison with that in the 1992 to 1993 period: clone A::1 remained dominant (47 versus 42.5%), whereas clone B::3 progressively declined (5 versus 35%) and clone C::5, the most susceptible to antibiotics, spread (44 versus 2.5%). Epidemiological investigations revealed that some clones had been introduced via patients transferred from other hospitals and that cross-infection occurred within and between wards. Major changes in the use of antibiotics, especially aminoglycosides, cyclines, and macrolides, likely played a role in the emergence and spread of GS-MRSA strains.
Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas
Authors: Shimoji Yoshihiro , Mori Yasuyuki , Hyakutake Koji , Sekizaki Tsutomu , Yokomizo Yuichi
Published: December 2019

We have previously described the creation by Tn
916
mutagenesis of avirulent transposition mutants from a highly virulent strain of
Erysipelothrix rhusiopathiae
, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn
916
insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of
E. rhusiopathiae
. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the
E. rhusiopathiae
chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of
Erysipelothrix
species and 27 strains of other genera different from
Erysipelothrix
. A 937-bp DNA fragment could be amplified from all
E. rhusiopathiae
strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify
E. rhusiopathiae
in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of
E. rhusiopathiae
was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 10
3
CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of
E. rhusiopathiae
in specimens.
Detection of Resistance to Amphotericin B among
Cryptococcus neoformans
Clinical Isolates: Performances of Three Different Media Assessed by Using E-Test and National Committee for Clinical Laboratory Standards M27-A Methodologies
Authors: Lozano-Chiu M. , Paetznick V. L. , Ghannoum M. A. , Rex J. H.
Published: December 2019

Although reliable detection of resistance in vitro is critical to the overall performance of any susceptibility testing method, the recently released National Committee for Clinical Laboratory Standards M27-A methodology for susceptibility testing of yeasts discriminates poorly between resistant and susceptible isolates of
Candida
spp. We have previously shown that both substitution of antibiotic medium 3 for RPMI 1640 medium in the microdilution variant of the M27-A method and use of the E-test agar diffusion methodology permit detection of amphotericin B-resistant
Candida
isolates. To determine the relevance of these observations to
Cryptococcus neoformans
, we have evaluated the performances of both the M27-A and the E-test methodologies with this yeast using three different media (RPMI 1640 medium, antibiotic medium 3, and yeast nitrogen base). As with
Candida
, we found that only antibiotic medium 3 permitted consistent detection of resistant isolates when testing was performed in broth by the M27-A method. When testing was performed by the E-test agar diffusion method, both RPMI 1640 medium and antibiotic medium 3 agar permitted ready detection of the resistant isolates. Reading of the results after 48 h of incubation was required for testing in broth by the M27-A method, while the MIC could be determined after either 48 or 72 h when the agar diffusion method was used.
Rapid Shell Vial Culture Technique for Detection of Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with Conventional Virus Isolation Method
Authors: Van Doornum G. J. J. , De Jong J. C.
Published: December 2019

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.
Direct Identification ofVibrio vulnificusin Clinical Specimens by Nested PCR
Authors: Lee Shee Eun , Kim Soo Young , Kim Sei Jong , Kim Hyun Soo , Shin Jong Hee , Choi Sang Ho , Chung Sun Sik , Rhee Joon Haeng
Published: December 2019

This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification ofVibrio vulnificusin clinical specimens. We designed two sets of primers targeting theV. vulnificushemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5′-GAC-TAT-CGC-ATC-AAC-AAC-CG-3′, and antisense, 5′-AGG-TAG-CGA-GTA-TTA-CTG-CC-3′, respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5′-GCT-ATT-TCA-CCG-CCG-CTC-AC-3′, and antisense, 5′-CCG-CAG-AGC-CGT-AAA-CCG-AA-3′, respectively) amplifies an internal 222-bp DNA fragment. We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA–0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. The DNA extraction method guaranteed sensitivity as well, even whenV. vulnificuscells were mixed with other bacteria such asEscherichia coliorStaphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU ofV. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18V. vulnificusculture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.
Outbreak of
Pseudomonas fluorescens
Bacteremia among Oncology Patients
Authors: Hsueh Po-Ren , Teng Lee-Jene , Pan Hui-Ju , Chen Yu-Chi , Sun Chun-Chuan , Ho Shen-Wu , Luh Kwen-Tay
Published: December 2019

From 7 to 24 March 1997, four patients developed
Pseudomonas fluorescens
bacteremia at the hospital; one on the oncology ward and the other three in the chemotherapy room. These patients all had underlying malignancies and had the Port-A-Cath (Smiths Industries Medical Systems, Deltec, Inc., St. Paul, Minn.) implants. Three patients had primary bacteremia, and one had Port-A-Cath-related infection. None of these patients had received a blood transfusion before the episodes of bacteremia. All patients recovered: two received antimicrobial agents with in vitro activity against the isolates, and the other two did not have any antibiotic treatment. A total of eight blood isolates were recovered from these patients during the febrile episodes that occurred several minutes after the infusion of chemotherapeutic agents via the Port-A-Cath. These isolates were initially identified as
P. fluorescens
or
Pseudomonas putida
(four),
Burkholderia
(
Ralstonia
)
pickettii
(three), and a non-glucose-fermenting gram-negative bacillus (one) by routine biochemical methods and the Vitek GNI card. These isolates were later identified as
P. fluorescens
on the basis of the characteristic cellular fatty acid chromatogram and the results of supplemental biochemical tests. The identification of identical antibiotypes by the E test and the random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates showed that the outbreak was caused by a single clone of
P. fluorescens
. Surveillance cultures of the possibly contaminated infusion fluids and disinfectants, which were performed 7 days after recognition of the last infected patient, failed to isolate
P. fluorescens
. This report of a small outbreak caused by
P. fluorescens
suggests that timely, accurate identification of unusual nosocomial pathogens is crucial for early initiation of an epidemiological investigation and timely control of an outbreak.
Molecular Genetic Variation in
Emmonsia crescens
and
Emmonsia parva
, Etiologic Agents of Adiaspiromycosis, and Their Phylogenetic Relationship to
Blastomyces dermatitidis
(
Ajellomyces dermatitidis
) and Other Systemic Fungal Pathogens
Authors: Peterson Stephen W. , Sigler Lynne
Published: December 2019

Emmonsia crescens
, an agent of adiaspiromycosis,
Blastomyces dermatitidis
, the agent of blastomycosis, and
Histoplasma capsulatum
, the agent of histoplasmosis, are known to form meiotic (sexual) stages in the ascomycete genus
Ajellomyces
(
Onygenaceae
,
Onygenales
), but no sexual stage is known for
E. parva
, the type species of the genus
Emmonsia
. To evaluate relationships among members of the putative
Ajellomyces
clade, large-subunit ribosomal and internal transcribed spacer region DNA sequences were determined from PCR-amplified DNA fragments. Sequences were analyzed phylogenetically to evaluate the genetic variation within the genus
Emmonsia
and evolutionary relationships to other taxa.
E. crescens
and
E. parva
are distinct species.
E. crescens
isolates are placed into two groups that correlate with their continents of origin. Considerable variation occurred among isolates previously classified as
E. parva
. Most isolates are placed into two closely related groups, but the remaining isolates, including some from human sources, are phylogenetically distinct and represent undescribed species. Strains of
B. dermatitidis
are a sister species of
E. parva
.
Paracoccidioides brasiliensis
and
Histoplasma capsulatum
are ancestral to most
Emmonsia
isolates, and
P. brasiliensis
, which has no known teleomorph, falls within the
Ajellomyces
clade.
Genetic and Serological Evidence for Multiple Instances of Unrecognized Transmission of Hepatitis C Virus in Hemodialysis Units
Authors: Mizuno Mari , Higuchi Terumi , Kanmatsuse Katsuo , Esumi Mariko
Published: December 2019

We investigated the unrecognized patient-to-patient transmission of hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1) of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA. A total of 24 HVR1 sequences, including the minor population of sequences of HCV isolates, from each patient were closely related and classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients were infected with HCV during an 18-month interval between examinations, and these HVR1 sequences fell into one of the five clusters. In another hemodialysis center, 5 of the 20 patients were HCV RNA positive, and two HVR1 sequences were found to be closely related and phylogenetically derived from the same cluster. The antibody responses of these patients to the HVR1 peptides representative of the genetic clusters revealed exactly the same clustering as that shown by phylogenetic analysis. These findings suggest that phylogenetic and serological analyses of HVR1 sensitively detect unrecognized and multiple transmission of HCV occurring within the same room in hemodialysis centers. Fingerprinting analyses using hypervariable regions of infectious agents are useful in identifying the precise route of transmission of infection.
Comparison of Restriction Enzyme Analysis, Arbitrarily Primed PCR, and Protein Profile Analysis Typing for Epidemiologic Investigation of an Ongoing
Clostridium difficile
Outbreak
Authors: Rafferty Mary Ellen , Baltch Aldona L. , Smith Raymond P. , Bopp Lawrence H. , Rheal Carol , Tenover Fred C. , Killgore George E. , Lyerly David M. , Wilkins Tracy D. , Schoonmaker Dianna J. , Hannett George E. , Shayegani Mehdi
Published: December 2019

During an outbreak of diarrhea in a general hospital in 1992, 166
Clostridium difficile
isolates from 102 patients were typed by restriction enzyme analysis (REA), arbitrarily primed PCR (AP-PCR), and protein profile analysis (PP) techniques. A total of 18 types and 5 subtypes were identified by REA, 32 types were identified by AP-PCR, and 9 types were identified by PP. Analysis of the data indicated the presence of a predominant strain among 76, 75, and 84% of the isolates by REA, AP-PCR, and PP, respectively. Subsequently, 45
C. difficile
isolates which had been collected in 1990 from 33 patients in the same hospital following a significant increase in the number of cases of diarrhea caused by
C. difficile
were studied by REA, AP-PCR, and PP typing techniques. Thirteen types and one subtype were identified by REA, 12 types were identified by AP-PCR, and 5 types were identified by PP. As with the isolates from 1992, a dominant strain was identified. This strain was represented by 53, 64, and 70% of the total number of isolates when the strains were typed by REA, AP-PCR, and PP, respectively. Every isolate (210 of 211) from both 1990 and 1992 that was available for typing was typeable by all three methods. Furthermore, the same dominant strain was identified in both 1990 and 1992 by each method. This study demonstrates that each of the three typing methods can be useful in epidemiologic investigations of
C. difficile
outbreaks and that one strain can be dominant in an institution over a number of years.
Assessment of Hepatitis C Virus Sequence Complexity by Electrophoretic Mobilities of Both Single-and Double-Stranded DNAs
Authors: Wang Yu-Ming , Ray Stuart C. , Laeyendecker Oliver , Ticehurst John R. , Thomas David L.
Published: December 2019

To assess genetic variation in hepatitis C virus (HCV) sequences accurately, we optimized a method for identifying distinct viral clones without determining the nucleotide sequence of each clone. Twelve serum samples were obtained from seven individuals soon after they acquired HCV during a prospective study, and a 452-bp fragment from the E2 region was amplified by reverse transcriptase PCR and cloned. Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined heteroduplex analysis (HDA) and a single-stranded conformational polymorphism (SSCP) method to determine the number of clonotypes (electrophoretically indistinguishable cloned cDNAs) as a measure of genetic complexity (this combined method is referred to herein as the HDA+SSCP method). We calculated Shannon entropy, incorporating the number and distribution of clonotypes into a single quantifier of complexity. These measures were evaluated for their correlation with nucleotide sequence diversity. Blinded analysis revealed that the sensitivity (ability to detect variants) and specificity (avoidance of false detection) of the HDA+SSCP method were very high. The genetic distance (mean ± standard deviation) between indistinguishable cloned cDNAs (intraclonotype diversity) was 0.6% ± 0.9%, and 98.7% of cDNAs differed by <2%, while the mean distance between cloned cDNAs with different patterns was 4.0% ± 3.2%. The sensitivity of the HDA+SSCP method compared favorably with either HDA or the SSCP method alone, which resulted in intraclonotype diversities of 1.6% ± 1.8% and 3.5% ± 3.4%, respectively. The number of clonotypes correlated strongly with genetic diversity (
R
2
, 0.93), but this correlation fell off sharply when fewer clones were assessed. This HDA+SSCP method accurately reflected nucleotide sequence diversity among a large number of viral cDNA clones, which should enhance analyses to determine the effects of viral diversity on HCV-associated disease. If sequence diversity becomes recognized as an important parameter for staging or monitoring of HCV infection, this method should be practical enough for use in laboratories that perform nucleic acid testing.
Comparison of PCR, Nested PCR, and Random Amplified Polymorphic DNA PCR for Detection and Typing of
Ureaplasma urealyticum
in Specimens from Pregnant Women
Authors: Knox Christine L. , Timms Peter
Published: December 2019

A PCR assay, using three primer pairs, was developed for the detection of
Ureaplasma urealyticum
, parvo biovar,
mba
types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5′ end and upstream control region of the
mba
gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for
mba
1, 262 bp for
mba
3, and 193 bp for
mba
6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested
mba
-type PCR described above. This nested PCR enabled the detection and typing of small numbers of
U. urealyticum
cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of
U. urealyticum
and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i)
U. urealyticum
is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii)
mba
type 6 was isolated significantly more often (
P
= 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.
Subcutaneous Hyalohyphomycosis Caused byColletotrichum gloeosporioides
Authors: Guarro Josep , Svidzinski Terezinha E. , Zaror Luís , Forjaz Maily H. , Gené Josepa , Fischman Olga
Published: December 2019

The coelomyceteColletotrichum gloeosporioideswas isolated in pure culture from subcutaneous nodules of the left forearm and elbow of a farmer after traumatic injury. To our knowledge, we report the first case involving this fungus as an etiological agent of subcutaneous infection. The in vitro inhibitory activities of amphotericin B, itraconazole, ketoconazole, miconazole, flucytosine, and fluconazole were studied.
Detection of
Aspergillus
Galactomannan: Comparison of an Enzyme-Linked Immunoassay and a Europium-Linked Time-Resolved Fluoroimmunoassay
Authors: Paugam A. , Sarfati J. , Romieu R. , Viguier M. , Dupouy-Camet J. , Latgé J. P.
Published: December 2019

With a view to improving the sensitivity of serological detection of
Aspergillus
galactomannan (GM), a europium-linked time-resolved fluoroimmunoassay was developed. This method was compared to an enzyme-linked immunosorbent assay using a peroxidase-conjugated detector antibody. No increase in the sensitivity of the detection of GM standards was seen with the europium-based fluoroimmunoassay.
Utility of Routine Testing of Bronchoalveolar Lavage Fluid for Cryptococcal Antigen
Authors: Kralovic Stephen M. , Rhodes Judith C.
Published: December 2019

All cryptococcal antigen (CrAg) testing performed at our institution between 1989 and 1994 was reviewed for utility of routinely testing of bronchoalveolar lavage fluid (BAL) for this antigen. Forty-two of 1,506 BAL specimens were positive. Seventeen of these were felt to represent false positives (sensitivity, 71%; positive predictive value, 0.59). The data on CrAg in cerebrospinal fluid and serum and the fungal culture and histological results of BAL specimens did not support continued, routine testing of BALs for CrAg to diagnose cryptococcosis.
PCR-Based Rapid Detection of
Mycobacterium tuberculosis
in Blood from Immunocompetent Patients with Pulmonary Tuberculosis
Authors: Ahmed Niyaz , Mohanty Ashok Kumar , Mukhopadhyay Utpal , Batish Virender Kumar , Grover Sunita
Published: December 2019

A PCR test based on insertion sequence IS1081 was developed to detect
Mycobacterium tuberculosis
complex organisms in the peripheral blood. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis. Seven of 16 (43.75%) blood samples were found to be positive for the circulating DNA copies of
M. tuberculosis
complex.
Extended-Spectrum β-Lactamase-Producing
Klebsiella pneumoniae
Strains Causing Nosocomial Outbreaks of Infection in the United Kingdom
Authors: Shannon Kevin , Stapleton Paul , Xiang Xiaoqin , Johnson Alan , Beattie Hamish , El Bakri Fatima , Cookson Barry , French Gary
Published: December 2019

Representative isolates from 10 distinct extended-spectrum β-lactamase-producing strains of
Klebsiella pneumoniae
that caused hospital outbreaks in the United Kingdom from 1991 to 1994 were examined for relationships between their enzymes and plasmids. The β-lactamases were identified by a combination of isoelectric focusing and gene sequencing. SHV-2 β-lactamase was produced by isolates from four outbreaks, SHV-5 was involved in three, and SHV-4, TEM-15, and TEM-26 were involved in one outbreak each. All of the extended-spectrum β-lactamases were encoded by self-transmissible plasmids, with sizes ranging from about 70 to 160 kb. No similarities between the restriction digest patterns of the extended-spectrum β-lactamase-encoding plasmids were detected, except to some extent between those that produced TEM-15 and TEM-26. Thus, outbreaks of hospital infection with these organisms in the United Kingdom from 1991 to 1994 involved distinct organisms and resistance plasmids and appeared to be unrelated.
Fluorescent Amplified-Fragment Length Polymorphism Analysis of an Outbreak of Group A Streptococcal Invasive Disease
Authors: Desai Meeta , Tanna Asha , Wall Robert , Efstratiou Androulla , George Robert , Stanley John
Published: December 2019

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was carried out for an outbreak of group A streptococcal (GAS) invasive disease. Streptococcal genomic DNAs were digested with endonucleasesEcoRI andMseI, site-specific adaptors were ligated, and PCR amplification was carried out with anEcoRI adaptor-specific primer labelled with fluorescent dye. Amplified fragments of up to 600 bp in size were separated on a polyacrylamide sequencing gel which contained internal size markers in each lane. These data were automatically scanned and analyzed, fragments were precisely sized (±1 bp), and electropherograms were generated for each genome with GeneScan 2.1 software. All isolates were compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP profiles, compared with 12 macrorestriction profiles by pulsed-field gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of invasive disease in a care-of-the-elderly system. It provided an automated analysis of the whole genome of bacterial isolates. It was reproducible, more discriminatory, and capable of higher throughput than other molecular typing methods. Given agreed conditions, FAFLP would be reproducible between laboratories for rapid characterization of outbreak strains.
Identification of
Legionella
Species by Random Amplified Polymorphic DNA Profiles
Authors: Lo Presti François , Riffard Serge , Vandenesch François , Etienne Jerome
Published: December 2019

Random amplified polymorphic DNA (RAPD) was used for the identification of
Legionella
species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42
Legionella
species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified
Legionella
isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of
Legionella
isolates to the species level without further restriction or hybridization.
Comparison of the MB/BacT System with a Revised Antibiotic Supplement Kit to the BACTEC 460 System for Detection of Mycobacteria in Clinical Specimens
Authors: Benjamin W. H. , Waites K. B. , Beverly A. , Gibbs L. , Waller M. , Nix S. , Moser S. A. , Willert M.
Published: December 2019

The MB/BacT system (MB/BacT) with a revised antibiotic supplement kit was compared with the BACTEC 460 system (BACTEC 460) in a test of 488 specimens submitted for mycobacterial culture from 302 patients. Twenty-four
Mycobacterium tuberculosis
isolates were detected by the BACTEC 460 versus 23 isolates by the MB/BacT. Mean time until detection of
M. tuberculosis
isolates identified by both systems was 11.9 days for the BACTEC 460 versus 13.7 days for the MB/BacT (
P
= 0.046).
M. avium
complex was detected in 12 specimens by the MB/BacT versus 10 specimens by the BACTEC 460. Only 8 of 14 (57%)
M. avium
isolates were detected by both systems, with a mean time until detection of 10.1 days for the BACTEC 460 and 14.2 days for the MB/BacT (
P
= 0.009). The BACTEC 460 and the MB/BacT detected
M. gordonae
in four specimens, but only a single specimen was positive by both systems. One
M. fortuitum
isolate and one of five
M. kansasii
isolates were recovered only by the BACTEC 460. The bacterial overgrowth rate was 7.0% for the MB/BacT versus 4.1% for the BACTEC 460. We found the MB/BacT to be comparable to the BACTEC 460 for mycobacterial detection. Even though time until detection with the MB/BacT was slightly longer (1.8 days longer for
M. tuberculosis
and 4.1 days for
M. avium
[mean values]) and the bacterial overgrowth rate was somewhat higher, the decreased labor, the availability of a computerized data management system, and the noninvasive, nonradiometric aspects of the MB/BacT offset these relative disadvantages and make it an acceptable alternative for use in the diagnostic laboratory.
Prevalence of
Porphyromonas gingivalis
and Periodontal Health Status
Authors: Griffen Ann L. , Becker Mitzi R. , Lyons Sharon R. , Moeschberger Melvin L. , Leys Eugene J.
Published: December 2019

Periodontitis is a common, progressive disease that eventually affects the majority of the population. The local destruction of periodontitis is believed to result from a bacterial infection of the gingival sulcus, and several clinical studies have provided evidence to implicate
Porphyromonas gingivalis
. If
P. gingivalis
is a periodontal pathogen, it would be expected to be present in most subjects with disease and rarely detected in subjects with good periodontal health. However, in most previous studies,
P. gingivalis
has not been detected in the majority of subjects with disease, and age-matched, periodontally healthy controls were not included for comparison. The purpose of the study reported here was to compare the prevalence of
P. gingivalis
in a group with periodontitis to that of a group that is periodontally healthy. A comprehensive sampling strategy and a sensitive PCR assay were used to maximize the likelihood of detection. The target sequence for
P. gingivalis
-specific amplification was the transcribed spacer region within the ribosomal operon.
P. gingivalis
was detected in only 25% (46 of 181) of the healthy subjects but was detected in 79% (103 of 130) of the periodontitis group (
P
< 0.0001). The odds ratio for being infected with
P. gingivalis
was 11.2 times greater in the periodontitis group than in the healthy group (95% confidence interval, 6.5 to 19.2). These data implicate
P. gingivalis
in the pathogenesis of periodontitis and suggest that
P. gingivalis
may not be a normal inhabitant of a periodontally healthy dentition.
Detection of Active Infection in Nonhuman Primates with Lyme Neuroborreliosis: Comparison of PCR, Culture, and a Bioassay
Authors: Pachner Andrew R. , Zhang Wei-Fen , Schaefer Henry , Schaefer Susan , O’Neill Tim
Published: December 2019

Ideally a diagnosis of infection of the central nervous system (CNS) is made by culture of the etiologic pathogen, but
Borrelia burgdorferi
, the causative agent of Lyme neuroborreliosis (LNB), is rarely cultured from the cerebrospinal fluid (CSF). PCR and measurement of specific antibody in the CSF also have their limitations. The role of available assays for LNB has not been studied carefully in a comparative investigation. There is a need to assess the reliability of assays and to increase the ability to document active infection in the CNS. The recent development of the nonhuman primate (NHP) model of LNB allowed us to address this need in a faithful model of human LNB. In this study we compared the abilities of PCR and culture to detect the presence of spirochetes in the CSF and brain tissue of infected NHPs and related these measures of infection to the development of anti-
B. burgdorferi
antibody. We also tested a bioassay, the mouse infectivity test (MIT), in this model. Fourteen of 16 CSFs from four NHPs were positive by at least one of these techniques. Detection of spirochetes in the CSF by PCR, the MIT, and culture was inversely related to the concomitant presence of anti-
B. burgdorferi
antibody intrathecally. The performance of any particular test was associated with the strength of the host immune response. In early CNS infection, when anti-
B. burgdorferi
antibody had not yet appeared, or in immunocompromised hosts, the MIT compared favorably to culture and PCR for infected NHPs; antibody in the CSF was the most useful assay for immunocompetent NHPs.
Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent
Authors: Kim Hyung-Yong , Rikihisa Yasuko
Published: December 2019

The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies (MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs recognized the HGE agent but not
Ehrlichia chaffeensis
,
Ehrlichia sennetsu
,
Ehrlichia canis
, or their host cells. MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates. Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and 5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection and for serodiagnosis.
Genetic Heterogeneity of Clinical Strains of
Yersinia enterocolitica
Traced by Ribotyping and Relationships between Ribotypes, Serotypes, and Biotypes
Authors: Lobato M. J. , Landeras E. , González-Hevia M. A. , Mendoza M. C.
Published: December 2019

A series of 74
Yersinia enterocolitica
clinical strains collected in a Spanish region and 10 reference strains, assigned to nine serotypes and five biotypes, were analyzed by ribotyping procedures. Riboprobing, performed separately with
Hin
dIII and
Bgl
I and using an
rrn
operon as the probe, generated 13 and 11 ribotypes (discrimination index [DI] = 0.56 and 0.55), respectively. PCR ribotyping, performed with primers complementary to conserved regions of 16S and 23S rRNA genes, generated 13 ribotypes (DI = 0.56). A combination of data from the three procedures allowed for further discrimination into 17 combined ribotypes (DI = 0.83). The dendrogram obtained by cluster analysis of data from riboprobing indicated a high heterogeneity of the ribosomal DNA regions of the strains under study (similarities between 10 and 92%), which were grouped into three clusters at a similarity level of 0.32. The major cluster included 10 branches, and 7 of these formed a subcluster (similarity coefficient, >83%) represented by strains of serotype O:3 and biotype 2, 3, or 4. The second cluster included four branches, represented by strains belonging to seven non-O:3 serotypes, biotypes 1A and 2, and two of these branches included pyrazinamidase-positive as well as pyrazinamidase-negative strains. The remaining three branches, represented by O:3–biotype 4 strains, formed a third cluster weakly related to the others. Data from this study showed that
Y. enterocolitica
O:3 organisms assigned to a prevalent and endemic lineage and non-O:3 organisms assigned to three other less-frequent lineages are circulating and causing human disease in the Spanish region under study.
Antigenemia in Patients with Paracoccidioidomycosis: Detection of the 87-Kilodalton Determinant during and after Antifungal Therapy
Authors: Gómez B. L. , Figueroa J. I. , Hamilton A. J. , Diez S. , Rojas M. , Tobón A. M. , Hay R. J. , Restrepo A.
Published: December 2019

Serological diagnosis and follow-up of paracoccidioidomycosis (PCM) patients have relied mainly on the detection of antibody responses by using techniques such as complement fixation (CF) and immunodiffusion. We recently described a novel inhibition enzyme-linked immunosorbent assay (inh-ELISA) which proved to be useful in the diagnosis of PCM via the detection of an 87-kDa determinant in patient sera (B. L. Gomez, J. I. Figueroa, A. J. Hamilton, B. Ortiz, M. A. Robledo, R. J. Hay, and A. Restrepo, J. Clin. Microbiol. 35:3278–3283, 1997). This test has now been assessed as a means of following up PCM patients. A total of 24 PCM patients, classified according to their clinical presentation (6 with the acute form of the disease, of whom two had AIDS, 12 with the multifocal form of the disease, and 6 with the unifocal form of the disease), were studied. The four human immunodeficiency virus-negative patients with acute PCM showed a statistically significant decrease in circulating antigen levels after the start of antifungal therapy. Antigen levels in this group became negative by our criteria (≤2.3 μg/ml) before week 20 and remained so in three of four of these patients. In contrast, the two AIDS patients who also presented with the acute form of PCM showed no statistically significant decrease in circulating antigen levels even after 68 weeks of therapy. Taken together as a group, the patients with the multifocal form showed a statistically significant decrease in antigenemia after 28 weeks of therapy. In addition, five of six patients with the unifocal form became antigen negative by week 40. Antigen level decrease mirrored clinical cure in the majority of patients in all clinical groups; in contrast, measurement of anti-PCM antibodies via the CF test showed wide fluctuations in titers during the follow-up period. The inh-ELISA for the detection of the 87-kDa
Paracoccidioides brasiliensis
determinant would appear to be a valuable additional tool in the follow-up of PCM patients.
Detection and Typing of
Borrelia burgdorferi
Sensu Lato in
Ixodes ricinus
Ticks Attached to Human Skin by PCR
Authors: Liebisch Gabriele , Sohns Bettina , Bautsch Wilfried
Published: December 2019

Live
Ixodes ricinus
ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR.
Borrelia
species were identified by 16S rRNA sequence analysis, which showed the presence of several species, some not yet defined, and a high prevalence of multiply infected ticks.
Comparison of the QUANTIPLEX HIV-1 RNA 2.0 Assay with the AMPLICOR HIV-1 MONITOR 1.0 Assay for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma of Patients Receiving Stavudine-Didanosine Combination Therapy
Authors: Segondy Michel , Izopet Jacques , Pellegrin Isabelle , Montes Brigitte , Dumon Beatrice , Pasquier Christophe , Peeters Martine , Fleury Herve J. A. , Puel Jacqueline , Reynes Jacques
Published: December 2019

We compared the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma in the Stadi trail, which evaluated a stavudine plus didanosine combination therapy in 52 patients. HIV-1 RNA baseline values measured with AMPLICOR HIV-1 MONITOR 1.0 were significantly higher than those measured with QUANTIPLEX HIV-1 RNA 2.0, and decreases in HIV-1 RNA levels from baseline were also found to be significantly higher when measured with the AMPLICOR HIV-1 MONITOR 1.0 assay. The frequency of HIV-1 RNA levels below the lower limit of quantitation was significantly higher with QUANTIPLEX HIV-1 RNA 2.0 than with AMPLICOR HIV-1 MONITOR 1.0. Reanalysis of these results by an ultrasensitive procedure of AMPLICOR HIV-1 MONITOR 1.0 or by a modified version of the test that included additional primers adapted for non-B HIV-1 clades yielded greater differences between the QUANTIPLEX HIV-1 RNA 2.0 assay and the AMPLICOR HIV-1 MONITOR 1.0 assay. Our results indicate that a valid comparison of the virological efficacies obtained with different antiretroviral drug regimens requires the use of the same viral load quantitation procedure; further standardization between the different HIV-1 RNA quantitation kits is therefore needed.
Determination of 16S rRNA Sequences of Enterococci and Application to Species Identification of Nonmotile
Enterococcus gallinarum
Isolates
Authors: Patel Robin , Piper Kerryl E. , Rouse Mark S. , Steckelberg James M. , Uhl Jim R. , Kohner Peggy , Hopkins Marlene K. , Cockerill Franklin R. , Kline Bruce C.
Published: December 2019

The 16S rRNA sequences of enterococcal species
E. faecium
,
E. faecalis
,
E. gallinarum
,
E. casseliflavus/flavescens
,
E. dispar
,
E. pseudoavium
,
E. sulfureus
,
E. malodoratus
,
E. raffinosus
,
E. cecorum
,
E. hirae
,
E. saccharolyticus
,
E. seriolicida
,
E. mundtii
,
E. avium
,
E. durans
,
E. columbae
, and
E. solitarius
are presented herein. These data were utilized to confirm the species identification of two nonmotile
E. gallinarum
isolates which had been previously phenotypically identified as
E. faecium
. The implications of this finding are discussed.
Effect of Oxygen on Growth of
Mycobacterium ulcerans
in the BACTEC System
Authors: Palomino J. C. , Obiang A. M. , Realini L. , Meyers W. M. , Portaels F.
Published: December 2019

The effect of low oxygen concentration on the growth of 15 strains of
Mycobacterium ulcerans
was evaluated in the BACTEC system. Reduced oxygen tension enhanced the growth of
M. ulcerans
, suggesting that this organism has a preference for microaerobic environments. Application of this observation may improve rates of isolation of
M. ulcerans
in primary culture from clinical samples and promote isolation of the bacterium from environmental sources.
Development of a Nested PCR for Detection of
Cryptococcus neoformans
in Cerebrospinal Fluid
Authors: Rappelli Paola , Are Riccardo , Casu Giuseppe , Fiori Pier Luigi , Cappuccinelli Piero , Aceti Antonio
Published: December 2019

We report the development of a nested-PCR-based assay for the detection of
Cryptococcus neoformans
in cerebrospinal fluid. The specificity and sensitivity of the test were assessed. The technique was then applied to 40 cerebrospinal fluid samples. We obtained positive reactions for all 21 clinical samples from patients who had been previously diagnosed as having cryptococcal meningitis by conventional techniques and negative reactions for all 19 negative controls. Nested PCR is here compared with other diagnostic methods currently used in patients’ follow-up exams during anticryptococcal therapy.
Comparison of Two Rapid Whole-Blood Tests for
Helicobacter pylori
Infection in Chinese Patients
Authors: Leung Wai Keung , Chan Francis K. L. , Falk Matthew S. , Suen Roamy , Sung Joseph J. Y.
Published: December 2019

Consecutive Chinese patients undergoing endoscopy for dyspepsia were tested for
Helicobacter pylori
infection by two rapid whole-blood tests: FlexPack HP (Abbott Laboratories) and Helisal One-Step (Cortecs Diagnostics). Biopsy-based tests (rapid urease test and histology) and the [
13
C]urea breath test were used as the “gold standard.” One hundred sixty-one consecutive patients were studied, and 88 (54.7%) were confirmed to have
H. pylori
infection. The sensitivities, specificities, and positive and negative predictive values were 81.8%, 83.6% (
P
= 0.008), 85.7% (
P
= 0.04), and 79.2% for FlexPack HP and 84.1%, 63.0% (
P
= 0.008), 73.3% (
P
= 0.047), and 76.7% for Helisal One-Step, respectively.