bluuebunny/crossref_metadata_2025 huggingface dataset:
Random Amplification of Polymorphic DNA and Microsatellite Genotyping of Pre- and Posttreatment Isolates of
Candida
spp. from Human Immunodeficiency Virus-Infected Patients on Different Fluconazole Regimens
Authors: Metzgar David , van Belkum Alex , Field Dawn , Haubrich Richard , Wills Christopher
Published: December 2019

Twelve patients infected with the human immunodeficiency virus (HIV) and with CD4 cell counts below 100 cells/μl received fluconazole daily (200 mg; five patients) or weekly (400 mg; seven patients) for fungal prophylaxis during a 6-month period. Oropharyngeal swabs were taken at regular intervals in order to detect colonization with
Candida
spp. All yeast isolates were examined with respect to the development over time of fluconazole resistance. Genetic diversity among the strains was assessed in order to discriminate between selection of a resistant subclone and patient recolonization. Genotyping was performed through random amplification of polymorphic DNA (RAPD) analysis. Specific site polymorphisms were assayed by tracking length variability in several microsatellite loci. Finally, to maximize resolution, one of these loci (
ERK1
) was analyzed by nucleotide sequencing. Although the number of strains analyzed was too small to allow statistical verification, it appeared that when fluconazole was given weekly, a smaller fraction of the strains showed diminished sensitivity than when it was given daily. Genetic analyses allowed three different scenarios to be discerned. Resistance development in an otherwise apparently unchanged strain was seen for 1 of the 12 patients. Clear strain replacement was observed for 3 of the remaining 11 patients. For all other patients minor differences were seen in either the RAPD genotype or the microsatellite allele composition during the course of treatment. In general, microsatellite sequence data is in agreement with data obtained by other methods, but occasionally within-patient heterogeneity is indicated. The present results show that during fluconazole treatment colonizing strains can remain identical, be replaced by clearly different strains, or undergo small changes. Within a patient there may be different levels of intrastrain variation.
Enzyme Immunoassay Detecting Teichoic and Lipoteichoic Acids versus Cerebrospinal Fluid Culture and Latex Agglutination for Diagnosis of
Streptococcus pneumoniae
Meningitis
Authors: Stuertz Kristin , Merx Imke , Eiffert Helmut , Schmutzhard Erich , Mäder Michael , Nau Roland
Published: December 2019

A newly developed enzyme immunoassay (EIA) was used to detect the presence of pneumococcal teichoic and lipoteichoic acids in cerebrospinal fluid (CSF) from patients with
Streptococcus pneumoniae
meningitis who were being treated with antibiotics. All initial CSF samples, which on culture grew
S. pneumoniae
, were positive in the EIA. A total of 14 subsequent culture-negative samples gave clear signals in the EIA up to day 15 after the onset of antibiotic treatment. For 11 CSF specimens, culture, microscopy, and latex agglutination were negative while the EIA detected pneumococcal antigens. The EIA did not react either with CSF of patients with meningitis caused by bacteria other than
S. pneumoniae
or by viral pathogens. In conclusion, this EIA can be a valuable tool for the diagnosis of
S. pneumoniae
meningitis from CSF samples in cases in which prior antimicrobial therapy minimizes the usefulness of culture or other antigen detection tests.
Specific Serological Diagnosis of Leprosy with a Recombinant
Mycobacterium leprae
Protein Purified from a Rapidly Growing Mycobacterial Host
Authors: Triccas James A. , Roche Paul W. , Britton Warwick J.
Published: December 2019

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the
Mycobacterium leprae
35-kDa protein, purified from the rapidly growing host
Mycobacterium smegmatis
, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.
Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis
Authors: Morata Pilar , Queipo-Ortuño Maria Isabel , de Dios Colmenero Juan
Published: December 2019

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 μg, thereby avoiding false-negative results.
Evidence of Nosocomial Infection in Japan Caused by High-Level Gentamicin-Resistant
Enterococcus faecalis
and Identification of the Pheromone-Responsive Conjugative Plasmid Encoding Gentamicin Resistance
Authors: Ma Xinghua , Kudo Michiaki , Takahashi Ayako , Tanimoto Koichi , Ike Yasuyoshi
Published: December 2019

A total of 1,799
Enterococcus faecalis
isolates were isolated from inpatients of Gunma University Hospital, Gunma, Japan, between 1992 and 1996. Four hundred thirty-two (22.3%) of the 1,799 isolates had high-level gentamicin resistance. Eighty-one of the 432 isolates were classified and were placed into four groups (group A through group D) with respect to the
Eco
RI restriction endonuclease profiles of the plasmid DNAs isolated from these strains. The 81 isolates were isolated from 36 patients. For 35 of the 36 patients, the same gentamicin-resistant isolates were isolated from the same or different specimens isolated from the same patient at different times during the hospitalization. For one other patient, two different groups of the isolates were isolated from the same specimen. Groups A, B, C, and D were isolated from 5, 14, 12, and 6 patients, respectively. The strains had multiple-drug resistance. The restriction endonuclease digestion patterns of the
E. faecalis
chromosomal DNAs isolated from isolates in the same group were also identical. The patients who had been infected with the gentamicin-resistant isolates from each group were geographically clustered on a ward(s). These results suggest that the isolates in each group were derived from a common source and had spread in the ward. The gentamicin-resistant isolates exhibited a clumping response upon exposure to pheromone (
E. faecalis
FA2-2 culture filtrate). The gentamicin resistance transferred at a high frequency to the recipient
E. faecalis
isolates by broth mating, and the pheromone-responsive plasmids encoding the gentamicin resistance were identified in these isolates.
Specific Differentiation between
Mycobacterium bovis
BCG and Virulent Strains of the
Mycobacterium tuberculosis
Complex
Authors: Magdalena Juana , Supply Philip , Locht Camille
Published: December 2019

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying
Mycobacterium bovis
BCG. The
senX3-regX3
IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the
senX3-regX3
IR, whereas all non-BCG
M. tuberculosis
complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other
M. tuberculosis
complex strains.
Improved Identification of Mycobacteria by Using the Microbial Identification System in Combination with Additional Trimethylsulfonium Hydroxide Pyrolysis
Authors: Müller K.-Dieter , Schmid Ernst N. , Kroppenstedt Reiner M.
Published: December 2019

The MIDI automated Microbial Identification System (MIS) uses gas chromatography (GC) analysis of whole-cell fatty acid methyl esters (FAMEs) between 9 and 20 carbons in length to characterize a wide range of bacterial genera and species, including mycobacteria. Mycolic acid cleavage products (MACPs) with chain lengths of C
22
to C
26
are not released by MIDI sample preparation of mycobacteria. Therefore, the MIS library search report often matches several mycobacterial species without any significant difference in the similarity indices. The problem is solved by adding trimethylsulfonium hydroxide (TMSH) instead of sodium sulfate in the last step of sample preparation, thus allowing the identification of MACPs in addition to FAMEs. Only one GC run parameter has to be changed: the temperature program must be extended from 260 to 310°C. The MIS library search report for the identification of bacteria is not disturbed by TMSH. The combination of conventional library search report with the information of typical MACP patterns yields significantly better discrimination of mycobacterial species than the MIDI method allows.
Persistence of Clones of Coagulase-Negative Staphylococci among Premature Neonates in Neonatal Intensive Care Units: Two-Center Study of Bacterial Genotyping and Patient Risk Factors
Authors: Vermont Clementien L. , Hartwig Nico G. , Fleer André , de Man Peter , Verbrugh Henri , van den Anker John , de Groot Ronald , van Belkum Alex
Published: December 2019

From 1 January 1995 until 1 January 1996, we studied the molecular epidemiology of blood isolates of coagulase-negative staphylococci (CoNS) in the Neonatal Intensive Care Units (NICUs) of the Sophia Children’s Hospital (SCH; Rotterdam, The Netherlands) and the Wilhelmina Children’s Hospital (WCH; Utrecht, The Netherlands). The main goal of the present study was to detect putatively endemic clones of CoNS persisting in these NICUs. Pulsed-field gel electrophoresis was used to detect the possible presence of endemic clones of clinical significance. In addition, clinical data of patients in the SCH were analyzed retrospectively to identify risk factors for the acquisition of positive blood cultures. In both centers, endemic CoNS clones were persistently present. Thirty-three percent of the bacterial isolates derived from blood cultures in the SCH belonged to a single genotype. In the WCH, 45% of all bacterial strains belonged to a single clone. These clones were clearly different from each other, which implies that site specificity is involved. Interestingly, we observe that the clonal type in the SCH differed significantly from the incidentally occurring strains with respect to both the average pH and partial CO
2
pressure of the patient’s blood at the time of bacterial culture. We found that the use of intravascular catheters, low gestational age, and a long hospital stay were important risk factors for the development of a putative CoNS infection. When the antibiotic susceptibility of the bacterial isolates was assessed, a clear correlation between the nature of the antibiotics most frequently used as a first line of defense versus the resistance profile was observed. We conclude that the intensive use of antibiotics in an NICU setting with highly susceptible patients causes selection of multiresistant clones of CoNS which subsequently become endemic.
Detection of a Novel Strain of Porcine Circovirus in Pigs with Postweaning Multisystemic Wasting Syndrome
Authors: Morozov Igor , Sirinarumitr Theerapol , Sorden Steven D. , Halbur Patrick G. , Morgan Marsha K. , Yoon Kyoung-Jin , Paul Prem S.
Published: December 2019

Swine infectious agents, especially viruses, are potential public health risks associated with the use of pig organs for xenotransplantation in humans. Therefore, there is a need for better characterization of swine viruses and for the development of diagnostic tests for their detection. We report here isolation of a novel strain of porcine circovirus (PCV) from pigs with postweaning multisystemic wasting syndrome (PMWS). Affected pigs exhibited severe interstitial pneumonia and lymphoid depletion. The complete nucleotide sequence (1,768 nucleotides) of the genome of the PCV isolate was determined and compared with the sequence of the PCV strain isolated from PK-15 cells. Sequence comparison revealed significant differences between the two PCV strains, with an overall DNA homology of 76%. Two major open reading frames (ORFs) were identified. ORF1 was more conserved between the two strains, with 83% nucleotide homology and 86% amino acid homology. ORF2 was more variable, with nucleotide homology of 67% and amino acid homology of 65%. PCR and in situ hybridization demonstrated abundant viral DNA in various organs of pigs with PMWS. In situ hybridization demonstrated that this strain of PCV targets multiple organs and infects macrophages, lymphocytes, endothelial cells, and epithelial cells.
Rapid and Specific Detection of ToxigenicStaphylococcus aureus: Use of Two Multiplex PCR Enzyme Immunoassays for Amplification and Hybridization of Staphylococcal Enterotoxin Genes, Exfoliative Toxin Genes, and Toxic Shock Syndrome Toxin 1 Gene
Authors: Becker Karsten , Roth Ricarda , Peters Georg
Published: December 2019

Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed forStaphylococcus aureusexotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA,entB,entC,entD, andentE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (etaandetb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of theS. aureustoxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel ofS. aureusreference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolatedS. aureusstrains for diagnostic purposes as well as research studies.
PCR Detection of North American and Central African Isolates of Epizootic Hemorrhagic Disease Virus (EHDV) Based on Genome Segment 10 of EHDV Serotype 1
Authors: Aradaib Imadeldin E. , Wilson William C. , Schore Caroline E. , Mohammed Mohammed E. H. , Yilma Tilahun D. , Cullor James S. , Osburn Bennie I.
Published: December 2019

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.
Clinical Evaluation of the ASTY Colorimetric Microdilution Panel for Antifungal Susceptibility Testing
Authors: Pfaller M. A. , Arikan S. , Lozano-Chiu M. , Chen Y.-S. , Coffman S. , Messer S. A. , Rennie R. , Sand C. , Heffner T. , Rex J. H. , Wang J. , Yamane N.
Published: December 2019

A method using a commercially prepared colorimetric microdilution panel (ASTY; Kyokuto Pharmaceutical Industrial Co., Ltd.) was compared in four different laboratories with the National Committee for Clinical Laboratory Standards (NCCLS) reference microdilution method by testing 802 clinical isolates of
Candida
spp. (
C. albicans
,
C. glabrata
,
C. tropicalis
,
C. parapsilosis
,
C. krusei
,
C. lusitaniae
,
C. guilliermondii
,
C. lipolytica
,
C. rugosa
, and
C. zeylanoides
) against amphotericin B, 5-fluorocytosine (5FC), fluconazole, and itraconazole. Reference MIC endpoints were established after 48 h of incubation, and ASTY endpoints were established after 24 and 48 h of incubation. ASTY endpoints were determined to be the time at which the color of the first well changed from red (indicating growth) to purple (indicating growth inhibition) or blue (indicating no growth). Excellent agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 93% at 24 h and 96% at 48 h. Agreement ranged from 90% with itraconazole and 5FC to 96% with amphotericin B at 24 h and from 92% with itraconazole to 99% with amphotericin B and 5FC at 48 h. The ASTY colorimetric microdilution panel method appears to be comparable to the NCCLS reference method for testing the susceptibilities of
Candida
spp. to a variety of antifungal agents.
Sequential Evaluation of Dogs Naturally Infected withEhrlichia canis,Ehrlichia chaffeensis,Ehrlichia equi,Ehrlichia ewingii, orBartonella vinsonii
Authors: Breitschwerdt Edward B. , Hegarty Barbara C. , Hancock Susan I.
Published: December 2019

Historically, disease manifestations in dogs seroreactive toEhrlichia canisantigens by indirect immunofluorescent antibody testing have been attributed to infection with eitherE. canisorEhrlichia ewingii. A 1996 study by Dawson and colleagues provided PCR evidence that healthy dogs from southeastern Virginia could be naturally infected withEhrlichia chaffeensis. This observation stimulated us to determine whichEhrlichiaspp. infected sick dogs that were referred to our hospital from the same region. Based upon PCR amplification with species-specific primers, sick dogs seroreactive toE. canisantigens were determined to be infected with fourEhrlichiaspecies:E. canis,E. chaffeensis,E. equi, andE. ewingii. Coinfection with threeEhrlichiaspecies (E. canis,E. ewingii, andE. equi) was documented for one dog. An additional canine pathogen presumed to be tick transmitted,Bartonella vinsoniisubsp.berkhoffii, was identified in 7 of 12 dogs. Importantly, our results indicate that in naturally infected dogs,E. chaffeensiscan cause severe disease manifestations that are clinically and serologically indistinguishable from disease manifestations ofE. canisorE. ewingii. In addition, our findings support the efficacy of doxycycline for treatment ofE. canis,E. equi, andE. ewingiiinfections but indicate that, based upon the persistence ofE. chaffeensisDNA for 1 year following treatment,E. chaffeensisinfection in dogs may be more refractory to doxycycline treatment. Undetected coinfection withBartonellamay also complicate the evaluation of treatment efficacy while resulting in disease manifestations that mimic ehrlichiosis.
Comparison of the Binax
Legionella
Urinary Antigen Enzyme Immunoassay (EIA) with the Biotest
Legionella
Urin Antigen EIA for Detection of
Legionella
Antigen in both Concentrated and Nonconcentrated Urine Samples
Authors: Domínguez J. A. , Galí N. , Pedroso P. , Fargas A. , Padilla E. , Manterola J. M. , Matas L.
Published: December 2019

We evaluated a newly commercial enzyme immunoassay (EIA) (Biotest
Legionella
Urin Antigen EIA; Biotest AG, Dreieich, Germany) for detection of antigens of all
Legionella pneumophila
serogroups with a relatively wide spectrum of cross-reactivity as well as antigens of other
Legionella
spp. by comparing its sensitivity and specificity with those of an EIA for detection of
L. pneumophila
serogroup 1 antigen (
Legionella
urinary antigen EIA; Binax, Portland, Maine). Both tests were performed with both concentrated and nonconcentrated urine samples. We also evaluated the capabilities of both EIAs to detect extracted soluble antigens of American Type Culture Collection (ATCC)
Legionella
strains (
L. pneumophila
serogroups 1 to 14,
L. bozemanii
, and
L. longbeachae
). The sensitivity of the Biotest EIA was 66.66% in nonconcentrated urine and 86.66% in concentrated urine. The sensitivity of the Binax EIA was 63.76% and 88.88% in nonconcentrated and concentrated urine, respectively. The specificity was 100% in nonconcentrated and concentrated urine for both assays. The Binax EIA and Biotest EIA detected extracted soluble antigens of
L. pneumophila
serogroups 1 to 14 and
L. bozemanii
ATCC strains. The cross-reactions observed with the Binax EIA were probably due to common epitopes directly related to lipopolysaccharide. Further studies are required to determine the usefulness of the Binax EIA for detection of urinary antigens from
Legionella
species and serogroups other than
L. pneumophila
serogroup 1. The Biotest EIA proved to be as rapid, sensitive, and specific as the Binax EIA for the diagnosis of legionellosis. Concentration of antigen present in urine increased the sensitivities of both techniques with no reduction in specificity.
Discrimination of
Burkholderia gladioli
from Other
Burkholderia
Species Detectable in Cystic Fibrosis Patients by PCR
Authors: Bauernfeind Adolf , Schneider Ines , Jungwirth Renate , Roller Carsten
Published: December 2019

A procedure for molecular identification of
Burkholderia gladioli
is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of
B. gladioli
from related species (
B. cepacia
,
B. multivorans
,
B. vietnamiensis
,
B. mallei
,
B. pseudomallei
,
Ralstonia pickettii
, and
R. eutropha
) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis.
Cord Formation in BACTEC Medium Is a Reliable, Rapid Method for Presumptive Identification of
Mycobacterium tuberculosis
Complex
Authors: McCarter Yvette S. , Ratkiewicz Irene N. , Robinson Ann
Published: December 2019

Serpentine cord formation in BACTEC 12B medium was evaluated as a rapid method for the presumptive identification of
M. tuberculosis
complex. Kinyoun acid-fast stained smears were prepared from 666 positive BACTEC 12B bottles and examined for the presence or absence of serpentine cording. Cord formation had a sensitivity, specificity, positive predictive value, and negative predictive value of 89.2, 99.2, 98.5, and 94.2%, respectively. The evaluation of the presence of cord formation in BACTEC 12B medium is reliable and permits the rapid presumptive reporting of
M. tuberculosis.
Detection of
Helicobacter pylori
in Stool Specimens by PCR and Antigen Enzyme Immunoassay
Authors: Makristathis Athanasios , Pasching Eva , Schütze Kurt , Wimmer Margit , Rotter Manfred L. , Hirschl Alexander M.
Published: December 2019

A highly sensitive seminested PCR assay to detect
Helicobacter pylori
DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for
H. pylori
detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom
H. pylori
had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.
Early Detection of
Mycobacterium tuberculosis
in BACTEC Cultures by Ligase Chain Reaction
Authors: Tortoli Enrico , Lavinia Federica , Simonetti M. Tullia
Published: December 2019

The LCx
Mycobacterium tuberculosis
ligase chain reaction system (Abbott Diagnostic Division, Abbott Park, Ill.) was used to detect
M. tuberculosis
in 150 consecutive BACTEC vials on the day on which a positive growth index (GI) was recorded. By LCx,
M. tuberculosis
DNA was detected in BACTEC vials on average 2.6 days before the presence of acid-fast bacilli could be confirmed by microscopic examination. A total of 106 of 108
M. tuberculosis
isolates were detected without centrifugation from bottles presenting very low GIs (average, 70; median, 33). No false-positive result was obtained from nontuberculous mycobacteria or from isolates with contaminants.
Diagnosis of
Penicillium marneffei
Infection by Quantitation of Urinary Antigen by Using an Enzyme Immunoassay
Authors: Desakorn V. , Smith M. D. , Walsh A. L. , Simpson A. J. H. , Sahassananda D. , Rajanuwong A. , Wuthiekanun V. , Howe P. , Angus B. J. , Suntharasamai P. , White N. J.
Published: December 2019

Penicillium marneffei
is a major cause of opportunistic infection in patients with AIDS in north and northeastern Thailand. A method for the quantitation of
P. marneffei
antigen in urine was developed by using fluorescein isothiocyanate-labelled purified rabbit hyperimmune immunoglobulin G in an enzyme-linked immunosorbent assay. This method was evaluated with 33 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects, 248 hospitalized patients without penicilliosis) from the same area in which penicilliosis is endemic. Urinary antigen was found in all 33 (100%) patients with penicilliosis, with a median titer of 1:20,480. With undiluted samples, 67 (27%) of 248 hospital patients and 3 (6%) of 52 healthy controls were reactive. At a cutoff titer of 1:40, the urine antigen detection assay had a diagnostic sensitivity of 97% and specificity of 98% (positive predictive value, 84%; negative predictive value, 99.7%). This test offers a valuable and rapid method for the diagnosis of penicilliosis in patients with AIDS and could be a useful addition to conventional diagnostic methods in areas in which penicilliosis is endemic.
New York 1 and Sin Nombre Viruses Are Serotypically Distinct Viruses Associated with Hantavirus Pulmonary Syndrome
Authors: Gavrilovskaya Irina , LaMonica Rachel , Fay Mary-Ellen , Hjelle Brian , Schmaljohn Connie , Shaw Robert , Mackow Erich R.
Published: December 2019

New York 1 virus (NY-1) and Sin Nombre virus (SN) are associated with hantavirus pulmonary syndrome (HPS). NY-1 and SN are derived from unique mammalian hosts and geographic locations but have similar G1 and G2 surface proteins (93 and 97% identical, respectively). Focus reduction neutralization assays were used to define the serotypic relationship between NY-1 and SN. Sera from NY-1-positive
Peromyscus leucopus
neutralized NY-1 and SN at titers of ≥1/3,200 and ≤1/400, respectively (
n
= 12). Conversely, SN-specific rodent sera neutralized NY-1 and SN at titers of <1/400 and 1/6,400, respectively (
n
= 13). Acute-phase serum from a New York HPS patient neutralized NY-1 (1/640) but not SN (<1/20), while sera from HPS patients from the southwestern United States had 4- to >16-fold-lower neutralizing titers to NY-1 than to SN. Reference sera to Hantaan, Seoul, and Prospect Hill viruses also failed to neutralize NY-1. These results indicate that SN and NY-1 define unique hantavirus serotypes and implicate the presence of additional HPS-associated hantavirus serotypes in the Americas.
Performance Characteristics of the BDProbeTec System for Direct Detection of
Mycobacterium tuberculosis
Complex in Respiratory Specimens
Authors: Pfyffer Gaby E. , Funke-Kissling Pascale , Rundler Eva , Weber Rainer
Published: December 2019

Strand displacement amplification (SDA) technology has been established in a fully automated system known as BDProbeTec. Target sequences of the insertion sequence IS
6110
and the 16S rRNA gene are simultaneously amplified, which thus allows the detection of
Mycobacterium tuberculosis
complex and, as an additional option, of most
Mycobacterium
species. Detection occurs via a chemiluminescent microwell assay that employs the simultaneous hybridization and capture of SDA products with a biotinylated capture probe and an alkaline phosphatase detector probe. We have evaluated the performance of the BDProbeTec system in detecting
M. tuberculosis
complex by testing 799 respiratory specimens and comparing the results to those obtained by conventional diagnostic techniques, i.e., microscopy and culture (solid and radiometric media).
M. tuberculosis
was cultivated from 41 specimens, of which 28 (68.4%) were smear positive and 13 (31.6%) were smear negative. The overall sensitivity of the SDA assay was 97.6% (for smear-positive specimens, 100%; for smear-negative specimens, 92.3%), and specificity was 95.0%. After resolution of the discrepancies by studying the patients’ clinical data, sensitivity and specificity were 97.9 and 96.5%, respectively, and positive and negative predictive values were 63.9 and 99.9%, respectively. These preliminary data demonstrate that the BDProbeTec system has promising performance characteristics with respiratory specimens and that it allows the detection of
M. tuberculosis
complex within hours.
Homogeneity of 16S-23S Ribosomal Intergenic Spacer Regions of
Tropheryma whippelii
in Swiss Patients with Whipple’s Disease
Authors: Hinrikson Hans Peter , Dutly Fabrizio , Altwegg Martin
Published: December 2019

The current genetic strategies used to identify
Tropheryma whippelii
, the putative agent of Whipple’s disease, are based on PCR-mediated amplification of a part of its 16S rRNA gene (16S rDNA). Because there is very little intraspecies variation in these molecules, they are not suitable as targets for epidemiologic investigations. However, the intergenic spacer region between the 16S and 23S rDNAs is usually much more variable and has repeatedly been used for epidemiologic purposes. We have therefore amplified the spacer region of
T. whippelii
directly from clinical specimens from nine independent Swiss patients with Whipple’s disease by PCR with primers complementary to the 3′ and 5′ ends of the 16S and 23S rDNAs, respectively. The amplicons were directly sequenced and the sequences were compared to the
T. whippelii
reference sequence in GenBank/EMBL (accession no.
X99636
). Complete sequence homogeneity was found between the samples from our nine patients; the spacer sequence was also identical to the reference sequence. However, the sequences corresponding to the 3′ and 5′ ends of the 16S and the 23S rDNAs of
T. whippelii
, respectively, differed from the respective sequences in GenBank/EMBL. The same sequence found in our patients was then found in a sample from the German patient from which the published sequence had been derived. We conclude that the 16S-23S rDNA spacer region seems to be very conserved in
T. whippelii
and that the respective reference entry in public databases should be revised.
Disseminated Infection Due to
Chrysosporium zonatum
in a Patient with Chronic Granulomatous Disease and Review of Non-
Aspergillus
Fungal Infections in Patients with This Disease
Authors: Roilides Emmanuel , Sigler Lynne , Bibashi Evangelia , Katsifa Helen , Flaris Nicolas , Panteliadis Christos
Published: December 2019

We report the first case of
Chrysosporium zonatum
infection in a 15-year-old male with chronic granulomatous disease who developed a lobar pneumonia and tibia osteomyelitis while on prophylaxis with gamma interferon. The fungus was isolated from sputum and affected bone, and hyphae were observed in the bone by histopathology. Therapy with amphotericin B eradicated the osteomyelitis and pneumonia, but pneumonia recurred in association with pericarditis and pleuritis during therapy with itraconazole. These manifestations subsided, and no recurrences occurred with liposomal amphotericin B therapy. Infections caused by
Chrysosporium
species are very rare, and
C. zonatum
has not previously been reported to cause mycosis in humans. This species, the anamorph of the heterothallic ascomycete
Uncinocarpus orissi
(family Onygenaceae), is distinguished by its thermotolerance, by colonies which darken from yellowish white to buff, and by club-shaped terminal aleurioconidia borne at the ends of short, typically curved stalks. The case isolate produced fertile ascomata in mating tests with representative isolates. The median (range) MICs for our isolate as well as those for two other human isolates and a nonhuman isolate determined by the National Committee for Clinical Laboratory Standards method adapted for moulds were ≤0.06 μg/ml (≤0.06 to 0.25 μg/ml) for amphotericin B, 0.687 μg/ml (0.25 to 2 μg/ml) for itraconazole, >128 μg/ml (>128 μg/ml) for flucytosine, and 48 μg/ml (32 to >128 μg/ml) for fluconazole.
Identification of
Yersinia
Species by the Vitek GNI Card
Authors: Linde Hans-Jörg , Neubauer Heinrich , Meyer Hermann , Aleksic Stojanca , Lehn Norbert
Published: December 2019

The Vitek GNI card was used to identify 212 isolates of 10
Yersinia
species. Identification was correct for 96.3% of the isolates (156 of 162) to the genus level and for 57.4% of the isolates (93 of 162) to the species level for
Yersinia
spp. listed in the Vitek database. We recommend additional identification methods for isolates assigned to the genus
Yersinia
by the Vitek system.
Definition of False-Positive Reactions in Screening for Hepatitis C Virus Antibodies
Authors: Schröter Matthias , Feucht Heinz-Hubert , Schäfer Peter , Zöllner Bernhard , Polywka Susanne , Laufs Rainer
Published: December 2019

The rate of false-positive hepatitis C virus enzyme immunoassay results was determined to be at least 10% among 1,814 reactive serum samples based on (i) negative results in an independent confirmation assay, (ii) negative PCR results, and (iii) no patients developing clinical or biochemical signs of hepatitis during a 1-year follow-up.
Evaluation of the Oxoid Dryspot Streptococcal Grouping Kit for Grouping Beta-Hemolytic Streptococci
Authors: Petts D. N.
Published: December 2019

The latex agglutination kits that are widely used for grouping of beta-hemolytic streptococci in clinical laboratories use liquid latex suspensions. The Oxoid Dryspot kit (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) uses predispensed latex dried onto reaction cards or cardboard strips. All streptococci of groups A (85 strains), B (87 strains), C (30 strains), D (38 strains), F (23 strains), and G (65 strains) were correctly grouped by using these reagents. The Oxoid Dryspot Streptococcal Grouping kit is a reliable method for grouping of the beta-hemolytic streptococci encountered in clinical laboratories.
Detection of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein p7 In Vitro and In Vivo
Authors: de Baar M. P. , van der Horn K. H. M. , Goudsmit J. , de Ronde A. , de Wolf F.
Published: December 2019

We developed and evaluated an immunoassay for the detection and quantification of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 using electrochemiluminescence technology. The assay had a dynamic range of 50 to 20,000 pg/ml and a lower detection limit equivalent to approximately 10
6.5
HIV-1 RNA copies/ml in culture supernatant. In vitro kinetic replication studies showed that the amount of p7 correlated strongly with the amount of p24 (
R
2
= 0.869;
P
< 0.0001) and viral RNA (
R
2
= 0.858;
P
= 0.0009). On the basis of the p7 and RNA concentrations, we calculated the median p7:RNA ratio to be approximately 1,400 p7 molecules per RNA molecule. HIV-1 p7 could be detected and quantified in culture supernatants of both group M subtype A to E viruses and group O viruses. The presence of p7 in vivo was evaluated in 81 serum samples collected from 62 HIV-1-infected individuals. Five samples were p7 positive, whereas 45 samples were HIV-1 p24 positive. Four of the five p7-positive samples were p24 positive as well. p7 could be detected only when serum HIV-1 RNA levels were greater than 10
6
copies/ml. Anti-p7 antibodies were found in six samples, and all six were p7 negative. In contrast to the in vitro results, it appeared that HIV-1 p7 could not be used as a marker for viral quantification in vivo, since more than 90% of the serum samples were p7 negative. In combination with the low prevalence of anti-p7 antibodies, this may, in turn, be advantageous: the p7 assay may be a good alternative to the p24 assay as the readout system for determination of neutralizing activity against HIV-1 in serum or other fluids containing anti-p24 antibodies.
Incidence of
Prevotella intermedia
and
Prevotella nigrescens
Carriage among Family Members with Subclinical Periodontal Disease
Authors: Fukui Katsuhito , Kato Naoki , Kato Haru , Watanabe Kunitomo , Tatematsu Norichika
Published: December 2019

We established a typing system for
Prevotella intermedia
and
Prevotella nigrescens
using the combination of PCR ribotyping and arbitrarily primed PCR (AP-PCR) fingerprinting and applied this system to the study of intrafamilial incidence of these species in the oral cavity. PCR ribotyping followed by subtyping by AP-PCR fingerprinting was applied to each type strain of
P. intermedia
and
P. nigrescens
and 54 isolates (32 isolates of
P. intermedia
and 24 isolates of
P. nigrescens
) from extraoral infections, resulting in an excellent discriminatory power (discrimination index, 0.99) for both species. A total of 18 subjects from six families, with the subjects from each family comprising the mother, the father, and a child who had subclinical early-stage to moderate adult periodontitis or simple gingivitis and who carried
P. intermedia
or
P. nigrescens
, or both, were enrolled in the study of intrafamilial carriage. When 20 colonies per specimen of subgingival plaque, if available, were picked from primary culture, 115
P. intermedia
and 178
P. nigrescens
isolates were recovered from the 18 subjects. Among the subjects studied, family members shared the same subtype strain(s) but non-family members did not. Multiple subtypes were found in 8 (57%) of the 14
P. nigrescens
-positive subjects but in only 3 (27%) of the 11
P. intermedia
-positive subjects; the difference was, however, not statistically significant (
P
= 0.14). These results suggest that the combination of PCR ribotyping and AP-PCR fingerprinting is well suited for the epidemiological study of
P. intermedia
and
P. nigrescens
and that each family seems to carry a distinct subtype(s) of these species.
Development of a Species-Specific PCR-Restriction Fragment Length Polymorphism Analysis Procedure for Identification of
Madurella mycetomatis
Authors: Ahmed Abdalla O. A. , Mukhtar Moawia M. , Kools-Sijmons Marly , Fahal Ahmed H. , de Hoog Sybren , van den Ende Bert Gerrits , Zijlstra Eduard E. , Verbrugh Henri , Abugroun El Sir A. M. , Elhassan Ahmed M. , van Belkum Alex
Published: December 2019

Madurella mycetomatis
is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of
M. mycetomatis
and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of
M. mycetomatis
reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of
M. mycetomatis
, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with
Cfo
I,
Hae
III,
Msp
I,
Sau
3AI,
Rsa
I, and
Spe
I restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.
Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis
Authors: Lucero Nidia E. , Foglia Luis , Ayala Sandra M. , Gall David , Nielsen Klaus
Published: December 2019

The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to
Brucella
is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the conventional tests. Further, the CELISA is simpler to perform that the CFT and may readily be standardized by the use of purified S-LPS antigen and monoclonal antibody for competition.
Australian Isolates of
Legionella longbeachae
Are Not a Clonal Population
Authors: Montanaro-Punzengruber J. C. , Hicks L. , Meyer W. , Gilbert G. L.
Published: December 2019

Legionella longbeachae
is almost as frequent a cause of legionellosis in Australia as
Legionella pneumophila
, but epidemiological investigation of possible environmental sources and clinical cases has been limited by the lack of a discriminatory subtyping method. The purpose of this study was to examine the genetic variability among Australian isolates of
L. longbeachae
serogroup 1. Pulsed-field gel electrophoresis (PFGE) of
Sfi
I fragments revealed three distinct pulsotypes among 57 clinical and 11 environmental isolates and the ATCC control strains of
L. longbeachae
serogroups 1 and 2. Each pulsotype differed by four bands, corresponding to <65% similarity. A clonal subgroup within each pulsotype was characterized by >88% similarity. The largest major cluster was pulsotype A, which included 43 clinical isolates and 9 environmental isolates and was divided into five subgroups. Pulsotypes B and C comprised smaller numbers of clinical and environmental isolates, which could each be further divided into three subgroups. The ATCC type strain of
L. longbeachae
serogroup 1 was classified as pulsotype B, subtype B3, while the ATCC type strain of
L. longbeachae
serogroup 2 was identified as a different pulsotype, LL2.
Sfi
I macrorestriction analysis followed by PFGE showed that the Australian
L. longbeachae
strains are not a single clonal population as previously reported.
Evidence from Molecular Fingerprinting of Limited Spread of Drug-Resistant Tuberculosis in Texas
Authors: Wilson Rebecca W. , Yang Zhenhua , Kelley Michael , Cave M. Donald , Pogoda Janice M. , Wallace Richard J. , Cegielski J. Peter , Dunbar Denise F. , Bergmire-Sweat David , Elliott L. Bruce , Barnes Peter F.
Published: December 2019

To determine the contribution of recent transmission to spread of drug-resistant tuberculosis in Texas, we performed IS
6110
-based and pTBN12-based restriction fragment length polymorphism (RFLP) analyses on
Mycobacterium tuberculosis
isolates. Isolates collected from 201 patients in Texas between 1992 and 1994 were studied. The distribution of cases was strikingly focal. All cases were reported from 35 of the 254 counties in Texas, and 74% (148 of 201) were reported from only 9 counties. One hundred sixty-one (80%) of the patients had
M. tuberculosis
isolates with unique RFLP patterns, and 41 (20%) patients were in 20 clusters, each comprising 2 to 3 patients. The largest number of cases of drug-resistant tuberculosis were reported in counties bordering Mexico, but the percentage of clustered cases was highest in northeast Texas and in counties that included the cities of Dallas, Fort Worth, and Houston. Compared to nonclustered patients, clustered patients were more likely to be African American and to have been born in the United States. Clustered patients were significantly more likely to be from the same geographic area, and clustered patients from the same geographic area were more likely to have isolates with identical drug susceptibility patterns, suggesting that they were linked by recent transmission. In 11 of 20 clusters, clustered patients were from geographically separate regions, and most isolates did not have identical drug susceptibility patterns, suggesting that tuberculosis was contracted from a common source in the remote past. Based on the low percentage of clustered cases and the small cluster size, we conclude that there is no evidence for the extensive transmission of drug-resistant tuberculosis in Texas.