bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Analysis of Non-O1, Non-O139
Vibrio cholerae
Associated with an Unusual Upsurge in the Incidence of Cholera-Like Disease in Calcutta, India
Authors: Sharma Charu , Thungapathra M. , Ghosh A. , Mukhopadhyay Asish K. , Basu Arnab , Mitra Rupak , Basu Indira , Bhattacharya S. K. , Shimada T. , Ramamurthy T. , Takeda T. , Yamasaki S. , Takeda Y. , Nair G. Balakrish
Published: December 2019

There was an inexplicable upsurge in the incidence of non-O1, non-O139
Vibrio cholerae
among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of
V. cholerae
isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139
V. cholerae
strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the
ctxA
,
zot
,
ace
, and
tcpA
genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of
Escherichia coli
and the various variants of these genes. Studies on the clonality of non-O1, non-O139
V. cholerae
strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (
hlyA
,
hlyU
,
hlx
,
toxR
, and
attRS1
) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of
V. cholerae
can cause diarrhea by a mechanism quite different from that of toxigenic
V. cholerae
O1 and O139, and we have proposed the nomenclature of enteropathogenic
V. cholerae
to include these serogroups.
Molecular Typing of
Borrelia burgdorferi
Sensu Lato by Randomly Amplified Polymorphic DNA Fingerprinting Analysis
Authors: Wang Guiqing , van Dam Alje P. , Spanjaard Lodewijk , Dankert Jacob
Published: December 2019

To study whether pathogenic clusters of
Borrelia burgdorferi
sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (
n
= 49) with various clinical entities and from ticks (
n
= 83) or dogs (
n
= 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species
B. burgdorferi
sensu stricto (
n
= 23),
Borrelia garinii
(
n
= 39),
Borrelia afzelii
(
n
= 59),
Borrelia japonica
(
n
= 1),
Borrelia valaisiana
(
n
= 12), and genomic group DN127 (
n
= 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of
B. burgdorferi
sensu stricto,
B. garinii
, and
B. afzelii
isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39
B. garinii
strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates.
B. afzelii
strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different
Borrelia
species as well as
Borrelia
isolates within species.
Staphylococcus lugdunensis
: Report of a Case of Peritonitis and an Easy-To-Perform Screening Strategy
Authors: Schnitzler Norbert , Meilicke Rainer , Conrads Georg , Frank Darius , Haase Gerhard
Published: December 2019

We report on a severe case of peritonitis due to
Staphylococcus lugdunensis
. The clinical course resembled an infection due to
S. aureus
more than one due to other coagulase-negative staphylococci. Therefore, we strongly recommend identification and propose an easy-to-perform procedure for screening of this pathogen.
Haemophilus parainfluenzae
Liver Abscess after Successful Liver Transplantation
Authors: Friedl J. , Stift A. , Berlakovich G. A. , Taucher S. , Gnant M. , Steininger R. , Mühlbacher F.
Published: December 2019

Haemophilus parainfluenzae
was isolated from a bile specimen and from an aspirate of a liver abscess in a 58-year-old liver-transplanted woman that was indicative of an invasion of the graft by an ascending route. Drug therapy, immunosuppression, rejection therapy, and Roux-en-Y choledochojejunostomy may have contributed to the septic course. Interdisciplinary cooperation was instrumental in diagnosis and successful management in this case.
Simplified Technique for Detection of Significant Bacteriuria by Microscopic Examination of Urine
Authors: Cardoso Celso Luíz , Muraro Carla Barbosa , Siqueira Vera Lúcia Dias , Guilhermetti Márcio
Published: December 2019

A comparative study of microscopic examination of 10 μl (simplified loop technique) and 50 μl (traditional drop technique) of uncentrifuged Gram-stained urine specimens for detection of significant bacteriuria was carried out. The results demonstrated that the 10-μl loop technique can be used as an alternative to the 50-μl drop technique for presumptive diagnosis of urinary-tract infection in bacteriological practice, with the advantages of greater rapidity and ease of performance.
Evaluation of the E Test for Antimicrobial Susceptibility Testing ofPseudomonas aeruginosaIsolates from Patients with Long-Term Bladder Catheterization
Authors: Di Bonaventura Giovanni , Ricci Evandro , Della Loggia Nicoletta , Catamo Giovanni , Piccolomini Raffaele
Published: December 2019

The E test was evaluated in comparison with reference agar methods (National Committee for Clinical Laboratory Standards) for the susceptibility testing of 248Pseudomonas aeruginosaisolates from bladder-catheterized patients against nine antibiotics. The E-test MICs correlated well with those determined by the agar dilution and disk diffusion reference methods (88 and 92.5% within 1 log2dilution step, respectively), confirming that the E test is a reliable method for the determination of MICs of antibiotics for catheterization-associatedP. aeruginosaisolates.
Enteroaggregative, Shiga Toxin-Producing
Escherichia coli
O111:H2 Associated with an Outbreak of Hemolytic-Uremic Syndrome
Authors: Morabito Stefano , Karch Helge , Mariani-Kurkdjian Patrizia , Schmidt Herbert , Minelli Fabio , Bingen Edouard , Caprioli Alfredo
Published: December 2019

Shiga toxin-producing
Escherichia coli
O111:H2 strains from an outbreak of hemolytic-uremic syndrome showed aggregative adhesion to HEp-2 cells and harbored large plasmids which hybridized with the enteroaggregative
E. coli
probe PCVD432. These strains present a novel combination of virulence factors and might be as pathogenic to humans as the classic enterohemorrhagic
E. coli.
Detection of Staphylococcal Superantigenic Toxins by a CD69-Specific Cytofluorimetric Assay Measuring T-Cell Activation
Authors: Lina Gerard , Cozon Grégoire , Ferrandiz Josette , Greenland Timothy , Vandenesch François , Etienne Jerome
Published: December 2019

The presence of staphylococcal superantigenic toxins in the supernatants of liquid cultures was detected by an easy and rapid method assessing the activation of T lymphocytes by cytofluorimetric measurement of CD69 expression.Staphylococcus aureuscells were grown in Eagle’s minimum essential medium supplemented with 5% heat-inactivated fetal calf serum. Supernatant fluids from allS. aureusstrains producing superantigen-related toxins, including enterotoxins A to E, toxic shock syndrome toxin, and exfoliative toxins A and B, induced CD69 expression in a significantly higher number of T cells than a cutoff of 2%. This CD69 assay might be used for initial detection of superantigens fromS. aureusstrains isolated in the context of staphylococcal toxemia or related chronic human diseases such as atopic dermatitis or Kawasaki syndrome.
Optimization of Specimen-Handling Procedures for Accurate Quantitation of Levels of Human Immunodeficiency Virus RNA in Plasma by Reverse Transcriptase PCR
Authors: Dickover Ruth E. , Herman Steven A. , Saddiq Khaliq , Wafer Deborah , Dillon Maryanne , Bryson Yvonne J.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma are currently widely used clinically for prognostication and in monitoring antiretroviral therapy. Accurate and reproducible results are critical for patient management. To determine the effects of specimen collection and handling procedures on quantitative measurement of HIV-1 RNA, we compared anticoagulants and sample processing times. Whole blood was collected from 20 HIV-1-infected patients in EDTA, acid citrate dextrose (ACD), and heparin tubes, aliquoted, and stored at room temperature. Plasma was separated from whole-blood aliquots prepared at ≤1, 3, 6, 24, and 48 h postcollection and then stored at −70°C until use. HIV-1 RNA levels were determined by the AMPLICOR HIV-1 MONITOR assay. Heparinized plasma samples, which were pretreated with heparinase prior to analysis, had the lowest baseline HIV-1 RNA levels. In the first 6 h, HIV-1 RNA levels decreased by 10, 20, and 31% in EDTA, ACD, and heparin tubes, respectively. From 6 to 48 h postcollection, HIV-1 RNA levels decreased in all anticoagulants, albeit at a slower, more consistent rate. Our results indicate that EDTA should be the anticoagulant of choice for plasma HIV-1 RNA measurement by reverse transcriptase PCR, but ACD tubes are acceptable if the plasma is separated within 6 h of blood collection. Caution must be applied in the interpretation of absolute HIV-1 RNA copy number values obtained with suboptimal specimen collection and processing procedures.
Spoligotyping Followed by Double-Repetitive-Element PCR as Rapid Alternative to IS
6110
Fingerprinting for Epidemiological Studies of Tuberculosis
Authors: Sola Christophe , Horgen Lionel , Maïsetti Jerome , Devallois Anne , Goh Khye Seng , Rastogi Nalin
Published: December 2019

A total of 129 clinical isolates of
Mycobacterium tuberculosis
representing 91 patients were typed by a combination of direct-repeat (DR)-based spoligotyping and an inter-IS
6110
–PGRS (polymorphic GC-rich region)–PCR, also designated double-repetitive-element PCR (DRE-PCR). During the first phase of this investigation, 72 clinical strains representing 52 patients were initially typed by IS
6110
-restriction fragment length polymorphism (RFLP) and DR-RFLP, followed by spoligotyping and DRE-PCR. In the second phase of this investigation, the discriminating ability of spoligotyping plus DRE-PCR was studied for 57 isolates from 39 patients who were suspected to be epidemiologically linked, and the typing results were later confirmed by IS
6110
-RFLP and DR-RFLP analyses. The molecular clustering of the isolates remained identical irrespective of the methods used. These results show that the association of two PCR-based fingerprinting techniques for molecular epidemiology of tuberculosis has a discriminating ability similar to the IS
6110
-RFLP reference method.
Phylogenetic Relationships among Highly Virulent Newcastle Disease Virus Isolates Obtained from Exotic Birds and Poultry from 1989 to 1996
Authors: Seal Bruce S. , King Daniel J. , Locke Devin P. , Senne Dennis A. , Jackwood Mark W.
Published: December 2019

Newcastle disease virus {NDV (avian paramyxovirus type 1 [APMV1])} isolates were recovered from imported exotic birds confiscated following importation into the United States, from waterbirds in the United States, and from poultry. The exotic birds probably originated from Central and South America, Asia, and Africa. The NDV isolates were initially characterized as highly virulent because of a short mean death time in embryonated chicken eggs. The isolates were typed as neurotropic or viscerotropic velogenic by intracloacal inoculation of adult chickens. Intracerebral pathogenicity index values for the virulent NDV isolates ranged from 1.54 to 1.90, compared to a possible maximum value of 2.0. These isolates had a dibasic amino acid motif in the fusion protein cleavage site sequence required for host systemic replication. Sequence differences were detected surrounding the fusion protein cleavage site and the matrix protein nuclear localization signal, indicating evolution of highly virulent NDV. Phylogenetically, these isolates were categorized with other highly virulent NDV strains that caused outbreaks in southern California poultry during 1972 and in cormorants in the north central United States and southern Canada during 1990 and 1992. These isolates are related to NDV that may have the APMV1 strain chicken/Australia/AV/32 or a related virus as a possible progenitor. Recent virulent NDV isolates and those recovered during disease outbreaks since the 1970s are phylogenetically distinct from current vaccine viruses and standard challenge strains.
Disseminated Invasive Infection Due to
Metarrhizium anisopliae
in an Immunocompromised Child
Authors: Burgner David , Eagles Gillian , Burgess Margaret , Procopis Peter , Rogers Maureen , Muir David , Pritchard Robert , Hocking Ailsa , Priest Michael
Published: December 2019

The first reported human case of possible disseminated infection with the insect pathogen
Metarrhizium anisopliae
var.
anisopliae
, a fungus which has been used commercially for biocontrol of insects, is described. The patient, a 9-year-old boy, had a 5-year history of pre-B-cell acute lymphoblastic leukemia and had been on chemotherapy throughout this period. After 10 days of profound neutropenia, lesions consistent with ecthyma gangrenosum appeared on his arms and legs.
M. anisopliae
was grown from specimens from three separate sites, collected at different times over a period of 1 month: a skin biopsy, a swab from the base of a lesion, and the core of another skin lesion which spontaneously discharged. The initial skin biopsy also showed histological evidence of epidermal necrosis and dermal invasion with fungal hyphae. A computed-tomography (CT) scan of the chest demonstrated a lesion in the superior segment of the lower lobe of the left lung. A CT scan of the brain revealed a lesion in the left temporoparietal region of the brain, consistent with an abscess. Despite antifungal treatment including liposomal amphotericin and 5-flucytosine, the patient eventually died. The initial portal of entry is unknown, but hematogenous dissemination to the skin appears likely because of the multiple ecthymic lesions, and the appearances of the brain lesion on the CT scan are consistent with a hematogenous fungal abscess.
Corneal Ulcer Caused by
Nocardia asteroides
in a Patient with Leprosy
Authors: Tendolkar Uma M. , Varaiya Ami , Ahuja A. S. , Motwane S. A. , Gogate A. S.
Published: December 2019

Nocardia asteroides
is a rare cause of keratitis usually associated with trauma. We report a case of corneal ulceration caused by
N. asteroides
in a patient with leprosy. This is the first case report of nocardial keratitis from Southeast Asia. The diminished corneal sensation in a patient with leprosy could be a predisposing factor for development or exacerbation of corneal ulceration.
Nosocomial Pneumonia Caused by Three Genetically Different Strains of
Legionella pneumophila
and Detection of These Strains in the Hospital Water Supply
Authors: Lück P. Christian , Wenchel Hans-Martin , Helbig Jürgen H.
Published: December 2019

A 44-year-old woman developed
Legionella pneumophila
pneumonia after cerebral surgery. Initially, one colony from a clinical specimen and two colonies from water samples, all belonging to serogroup 12, did not match when their DNA restriction patterns were compared. When additional colonies from the water specimens were analyzed, a serogroup 12 strain complementary to that found in the clinical specimen was identified. Other colonies from the clinical specimen were identified as serogroup 12 strains complementary to those identified from the water. In addition, the same serogroup 1 strain was isolated from the patient and the water system.
Identification and Purification of Specific
Penicillium marneffei
Antigens and Their Recognition by Human Immune Sera
Authors: Jeavons L. , Hamilton A. J. , Vanittanakom N. , Ungpakorn R. , Evans E. G. V. , Sirisanthana T. , Hay R. J.
Published: December 2019

Disseminated infection with the dimorphic pathogenic fungus
Penicillium marneffei
is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from
P. marneffei
-infected patients (
n
= 21) to develop Western blots of
P. marneffei
cytoplasmic yeast antigen (CYA).
P. marneffei
CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with
P. marneffei
infection and with pooled sera from patients with aspergillosis (
n
= 20), candidiasis (
n
= 10), cryptococcosis (
n
= 9), and histoplasmosis (
n
= 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the
P. marneffei
-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of
P. marneffei
-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from
P. marneffei
and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with
P. marneffei.
Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children
Authors: Matar Ghassan M. , Sidani Nada , Fayad Michel , Hadi Usamah
Published: December 2019

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included
Haemophilus
,
Streptococcus
, and
Moraxella
(
Branhamella
)
catarrhalis
, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) a
Haemophilus
-specific probe (probe RDR125) as a primer along with DG74, (ii) a
Streptococcus
-specific primer (primer STR1; designed by us) along with DG74, and (iii) an
M. catarrhalis
-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive for
Haemophilus
, 3 (8.6%) were positive for
Streptococcus
, and 10 (28.6%) were positive for
M. catarrhalis
. Ten samples (28.6%) exhibited a mixed infection and were positive for both
Haemophilus
and
M. catarrhalis
. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.
Use of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide for Rapid Detection of Rifampin-ResistantMycobacterium tuberculosis
Authors: Mshana Robert N. , Tadesse Genet , Abate Getahun , Miörner Håkan
Published: December 2019

We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-culturedMycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitiveM. bovis(BCG) andM. tuberculosisexposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistantM. tuberculosisthat is affordable even by low-income countries where tuberculosis is a major public health problem.
Detection of Kanamycin-Resistant
Mycobacterium tuberculosis
by Identifying Mutations in the 16S rRNA Gene
Authors: Suzuki Yasuhiko , Katsukawa Chihiro , Tamaru Aki , Abe Chiyoji , Makino Masanao , Mizuguchi Yasuo , Taniguchi Hatsumi
Published: December 2019

In
Mycobacterium smegmatis
and a limited number of
Mycobacterium tuberculosis
strains, the involvement of alterations of the 16S rRNA gene (
rrs
) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the
rrs
gene and the kanamycin-resistant phenotype in clinically isolated
M. tuberculosis
strains were correlated, 43 kanamycin-resistant strains (MICs, ≧200 μg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the
rrs
gene and the intervening sequence between the
rrs
gene and 23S rRNA (
rrl
) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases
Tai
I and
Tsp
45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease,
Bst
UI or
Dde
I.
Typing of
Helicobacter pylori vacA
Gene and Detection of
cagA
Gene by PCR and Reverse Hybridization
Authors: van Doorn L. J. , Figueiredo C. , Rossau R. , Jannes G. , van Asbroeck M. , Sousa J. C. , Carneiro F. , Quint W. G. V.
Published: December 2019

The present report describes an analysis of two virulence genes of
Helicobacter pylori
. Parts of the
cagA
gene, as well as parts from the signal (s) and middle (m) regions of the mosaic
vacA
gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the
vacA
s region (s1a, s1b, and s2 alleles), the
vacA
m region (m1 and m2 alleles), and the
cagA
gene. A total of 103
H. pylori
-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (
n
= 55) and The Netherlands (
n
= 48) were tested by the PCR-LiPA.
cagA
was detected in 84 and 73% of the Portuguese and Dutch patients, respectively.
vacA
typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type s1b, whereas most Dutch patients (61%) contained type s1a (
P
< 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (
P
= 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of
vacA
type s1 (s1a or s1b;
P
= 0.008) and
cagA
(
P
= 0.003) genes.
Molecular Epidemiological Study of
Aspergillus fumigatus
in a Bone Marrow Transplantation Unit by PCR Amplification of Ribosomal Intergenic Spacer Sequences
Authors: Radford Sarah A. , Johnson Elizabeth M. , Leeming John P. , Millar Michael R. , Cornish Jacqueline M. , Foot Annabel B. M. , Warnock David W.
Published: December 2019

We have developed a PCR-based method for the subspecific discrimination of
Aspergillus fumigatus
types by using two primers designed to amplify the intergenic spacer regions between ribosomal DNA transcription units. The method permitted the reproducible discrimination of 11 distinct DNA types among a total of 119 isolates of
A. fumigatus
collected from patients and from the environment of a bone marrow transplantation (BMT) unit over a three-year period. Ten DNA types of
A. fumigatus
were isolated from patients in the BMT unit; eight of these types were also found in the hospital environment, and six of these were present in the unit itself. Thirteen BMT patients developed infection with one of three DNA types some months after these had first been found in the environment of the unit. In other instances, the same DNA types of
A. fumigatus
were isolated from BMT patients that were later recovered from the environment of the unit. Several DNA types of
A. fumigatus
were found in the hospital environment over an 18-month period. Molecular typing of multiple isolates of
A. fumigatus
, obtained from postmortem tissue samples, showed that one patient was infected with a single DNA type, but two others had up to three different DNA types. Our findings suggest that
A. fumigatus
infection in BMT recipients may be nosocomial in origin and underline the need for careful environmental monitoring of units in which high-risk patients are housed.
Persistence of a Multidrug-Resistant
Pseudomonas aeruginosa
Clone in an Intensive Care Burn Unit
Authors: Hsueh Po-Ren , Teng Lee-Jene , Yang Pan-Chyr , Chen Yu-Chi , Ho Shen-Wu , Luh Kwen-Tay
Published: December 2019

Long-term colonization of various body sites with a multidrug-resistant
Pseudomonas aeruginosa
clone (resistant to piperacillin, cefoperazone, ceftazidime, aztreonam, imipenem, cefepime, cefpirome, ofloxacin, ciprofloxacin, minocycline, and aminoglycosides) with subsequent severe infections in burn patients has not been reported previously. Thirty-nine isolates of multidrug-resistant
P. aeruginosa
(resistant to ceftazidime and at least three of the agents listed above) recovered from various clinical samples from three patients in an intensive care burn unit from April 1997 to May 1997 and seven preserved isolates recovered from six patients in other medical wards at National Taiwan University Hospital from April 1996 to May 1997 were studied for their epidemiological relatedness. The epidemic could be attributed to a multidrug-resistant
P. aeruginosa
clone belonging to serogroup O:F (serogroup O:4) by means of antimicrobial susceptibility testing, O serogrouping, and analysis of the randomly amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates. The epidemic strain persisted in the three patients for weeks to months; in the meantime, these patients had received multiple antimicrobial agents for the management of intervening episodes of invasive infections (bacteremia, ventilator-associated pneumonia, and/or catheter-related sepsis) caused by this strain, as well as concomitant infections due to other organisms. The strain had been isolated only once previously, from a burn patient who was on the unit in December 1996. The present report, describing a small outbreak due to
P. aeruginosa
, documents the fact that a single clone of multidrug-resistant
P. aeruginosa
can cause long-term persistence in different body sites of burn patients and that the colonization can subsequently result in various severe infections.
Reverse Transcription-PCR Detection of
Mycobacterium leprae
in Clinical Specimens
Authors: Kurabachew Mekonnen , Wondimu Assefa , Ryon Judith J.
Published: December 2019

A reverse transcription (RT)-PCR assay targeting the 16S rRNA of
Mycobacterium leprae
was developed to detect the organism in clinical specimens. A 171-bp fragment was amplified when
M. leprae
RNA was used as a template but not when a panel of RNAs from 28 potentially cross-reacting mycobacterial species, seven genera related to
Mycobacterium
, and three organisms normally found among skin or nose flora were tested. As few as 10 organisms isolated from infected tissue could be detected, confirming the sensitivity of the assay. When the test was applied to clinical specimens,
M. leprae
was detected in 82% of skin biopsy specimens obtained from untreated leprosy patients, while skin biopsy specimens from healthy volunteers and patients with other dermatological disorders were negative. The sensitivity of the RT-PCR was higher than that of slit skin smear staining for acid-fast bacilli or acid-fast staining of fixed biopsy specimens since 53% of acid-fast bacillus-negative biopsy specimens were RT-PCR positive. Because 16S rRNA is rapidly degraded upon cell death, the assay may detect only viable organisms and may prove to be useful in assessing the efficacy of chemotherapy.
Identification of Acinetobacters on Blood Agar in Presence of
d
-Glucose by Unique Browning Effect
Authors: Siau Hong , Yuen Kwok-Yung , Ho Pak-Leung , Luk Wei-Kwang , Wong Samson S. Y. , Woo Patrick C. Y. , Lee Rodney A. , Hui Wai-Ting
Published: December 2019

A positive phenotypic characteristic of glucose-oxidizing acinetobacters was demonstrated with blood agar containing
d
-glucose. Glucose-oxidizing
Acinetobacter baumannii
,
Acinetobacter
genospecies 3,
Acinetobacter lwoffii
, and
Acinetobacter
genospecies 13 sensu Tjernberg and Ursing caused a unique brown discoloration of media supplemented with 5% blood (of horse, sheep, or human origin) and an aldose sugar (0.22 M
d
-glucose,
d
-galactose,
d
-mannose,
d
-xylose, or lactose). The browning effect was not observed when a ketose sugar (
d
-fructose or sucrose) was substituted for the aldose sugar or under high osmolarity in the presence of mannitol, glycerol, or sodium chloride. Other gram-negative nonfermenters (non-glucose-oxidizing acinetobacters,
Pseudomonas aeruginosa
, other
Pseudomonas
spp.,
Stenotrophomonas maltophilia
,
Flavobacterium
spp., and
Moraxella
spp.) did not cause similar discoloration. This novel browning effect may serve as an alternative trait for identifying glucose-oxidizing acinetobacters.
Nonrandom Association of IS6110andMycobacterium tuberculosis: Implications for Molecular Epidemiological Studies
Authors: McHugh T. D. , Gillespie S. H.
Published: December 2019

IS6110restriction fragment length polymorphism typing is now established as the primary typing method forMycobacterium tuberculosis. It has been assumed that the position of bands is random. Thus, the discrimination of the technique increases in proportion to the copy number. Two collections ofM. tuberculosiswere investigated to test this hypothesis. We identified 33 positions in isolates from a Tanzanian collection and 25 positions in isolates from a London, United Kingdom, collection where bands were significantly more likely to be present than would be expected by chance. These data suggest that band position is not random, and this possibility may have an impact on the interpretation of molecular epidemiological studies ofM. tuberculosis.
Simplification of a Locus-Specific DNA Typing Method (Vir Typing) for
Streptococcus pyogenes
Authors: Hartas J. , Hibble M. , Sriprakash K. S.
Published: December 2019

We describe a simplification of a highly discriminatory molecular typing method, called Vir typing, for
Streptococcus pyogenes
(D. Gardiner, J. Hartas, B. Currie, J. D. Mathews, D. J. Kemp, and K. S. Sriprakash, PCR Methods Appl. 4:288–293, 1995). The procedure can be completed within a day, is reproducible, and can be applied directly to colonies growing on primary culture plates, allowing rapid establishment of strain identity in an outbreak.
Association of
Providencia alcalifaciens
with Diarrhea in Children
Authors: Albert M. John , Faruque A. S. G. , Mahalanabis D.
Published: December 2019

It has been demonstrated in previous studies that
Providencia alcalifaciens
can produce diarrhea by an invasive mechanism. In the present study,
P. alcalifaciens
was isolated from the stool specimens of 17 of 814 diarrheal children younger than 5 years of age (2.1%) and from those of 4 of 814 matched controls (0.49%) (
P
= 0.004), indicating that the organism is significantly associated with diarrhea. However, 71% of
P. alcalifaciens
-positive diarrheal children had simultaneous infections with other recognized enteric pathogens.
Isolation of Influenza C Virus during an Outbreak of Influenza A and B Viruses
Authors: Greenbaum Evgenia , Morag Avraham , Zakay-Rones Zichria
Published: December 2019

During the winter of 1996 to 1997 two cases of influenza C were confirmed, one by isolation and the second by serology (fourfold increase in hemagglutination inhibition antibodies). The cases of influenza C occurred during an outbreak of influenza A (H
3
N
2
) and B viruses. The positive isolation was from one of three throat washings sent to the laboratory, and the other case was from a group of 51 students participating in a study of influenza virus vaccination. It seems, therefore, that influenza C virus should also be considered when examining patients with respiratory infections during the influenza season.
Number of Days Required for Recovery of Mycobacteria from Blood and Other Samples
Authors: Esteban Jaime , Molleja Ana , Fernández-Roblas Ricardo , Soriano Francisco
Published: December 2019

From 1991 to 1996, 541 blood samples were tested for the presence of mycobacteria; 56 were positive (30 patients, 26 human immunodeficiency virus positive). The species found were
Mycobacterium avium
(41 samples from 18 patients),
Mycobacterium tuberculosis
(12 samples from 9 patients), and three other species (1 sample each). The average time to detection was 25.23 days (22.65 for
M. avium
and 35.33 for
M. tuberculosis
). For 10 patients, the blood isolate was the only mycobacterium detected (4
M. tuberculosis
).
Immunodiagnosis of Human Granulocytic Ehrlichiosis by Using Culture-Derived Human Isolates
Authors: Ravyn M. Dana , Goodman Jesse L. , Kodner Carrie B. , Westad Deborah K. , Coleman Lisa A. , Engstrom Suzanne M. , Nelson Curt M. , Johnson Russell C.
Published: December 2019

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by anEhrlichiaspecies closely related toEhrlichia equiandEhrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused byEhrlichia chaffeensis,Rickettsia rickettsii, andCoxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples fromR. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests forBorrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n= 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure toEhrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.
Comparison of Performances of Two Commercially Available Tests, a PCR Assay and a Ligase Chain Reaction Test, in Detection of Urogenital
Chlamydia trachomatis
Infection
Authors: Puolakkainen Mirja , Hiltunen-Back Eija , Reunala Timo , Suhonen Satu , Lähteenmäki Pekka , Lehtinen Matti , Paavonen Jorma
Published: December 2019

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx
Chlamydia trachomatis
assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland.
Chlamydia trachomatis
was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of
C. trachomatis
infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital
C. trachomatis
infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic
C. trachomatis
infections.
Hospital Specificity, Region Specificity, and Fluconazole Resistance of
Candida albicans
Bloodstream Isolates
Authors: Pfaller M. A. , Lockhart S. R. , Pujol C. , Swails-Wenger J. A. , Messer S. A. , Edmond M. B. , Jones R. N. , Wenzel R. P. , Soll D. R.
Published: December 2019

In a survey of bloodstream infection (BSI) isolates across the continental United States, 162
Candida albicans
isolates were fingerprinted with the species-specific probe Ca3 and the patterns were analyzed for relatedness with a computer-assisted system. The results demonstrate that particular BSI strains are more highly concentrated in particular geographic locales and that established BSI strains are endemic in some, but not all, hospitals in the study and undergo microevolution in hospital settings. The results, however, indicate no close genetic relationship among fluconazole-resistant BSI isolates in the collection, either from the same geographic locale or the same hospital. This study represents the first of three fingerprinting studies designed to analyze the origin, genetic relatedness, and drug resistance of
Candida
isolates responsible for BSI.
Identification of New Influenza B Virus Variants by Multiplex Reverse Transcription-PCR and the Heteroduplex Mobility Assay
Authors: Zou Shimian , Stansfield Carol , Bridge Jodi
Published: December 2019

A quick genetic approach for the screening of influenza virus variants was developed in this laboratory (S. Zou, J. Clin. Microbiol. 35:2623–2627, 1997). It uses multiplex reverse transcription and multiplex PCR to amplify and differentiate the variable region of the hemagglutinin genes of different types and subtypes of influenza viruses. Variants within the same type or subtype are then identified by the heteroduplex mobility shift assay of the amplicons. The method was used to screen influenza virus isolates received from provincial laboratories during the 1996–1997 season and was able to identify new influenza B virus variants. Sequencing of the amplicons derived from the hemagglutinin gene of the identified variants and comparison with the vaccine strain B/Harbin/7/94 showed substitution rates of 2.26 to 2.55% at the nucleotide level and 4.26 to 4.68% at the amino acid level. The result further demonstrated that the approach provides a quick, sensitive, and reliable screening for influenza virus variants. It also suggested the necessity of close monitoring of influenza B virus isolates in the 1997–1998 season and critical evaluation of the reference strain for the type B influenza virus.
Multicenter Comparison of BACTEC 9050 and BACTEC 9240 Blood Culture Systems
Authors: Murray Patrick R. , Hollick Gary E. , Jerris Robert C. , Wilson Michael L.
Published: December 2019

The overall recovery of organisms and time to detection with the BACTEC 9050 and BACTEC 9240 systems were compared in a multicenter evaluation. In the first phase of the study, a total of 4,383 compliant aerobic (Plus Aerobic/F) blood culture sets were processed. There was no significant difference in the recovery of individual groups of organisms with the two systems, with the exception of
Streptococcus pneumoniae
which was isolated more frequently with BACTEC 9050. False-positive signals occurred more often with BACTEC 9240 (58 cultures) than with BACTEC 9050 (43 cultures), but false-negative cultures were uncommon with both systems (3 cultures for each system). Time to detection of positive cultures of clinically significant organisms was essentially the same with both instruments. In the second phase of the study, 2,431 compliant anaerobic (Plus Anaerobic/F) blood culture sets were processed. There was no significant difference in the recovery of organisms with BACTEC 9050 compared with BACTEC 9240. Significantly (
P
< 0.03) more false-positive signals occurred with BACTEC 9240 (15 cultures) than with BACTEC 9050 (4 cultures). Likewise, more false-negative cultures occurred with BACTEC 9240 (11 cultures) than with BACTEC 9050 (8 cultures). Time to detection of positive cultures of clinically significant organisms was essentially the same with both systems with the exception of anaerobes (
N
= 10), which were recovered earlier (
P
< 0.01) with BACTEC 9240 (35.0 h) than with BACTEC 9050 (61.4 h).
Detection of Antigen in Sera of Patients with Invasive Aspergillosis: Intra- and Interlaboratory Reproducibility
Authors: Verweij Paul E. , Erjavec Zoran , Sluiters Wim , Goessens Wil , Rozenberg-Arska Marja , Debets-Ossenkopp Yvette J. , Guiot Henri F. L. , Meis Jacques F. G. M.
Published: December 2019

The intra- and interlaboratory reproducibilities of a commercial sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of
Aspergillus
galactomannan in serum (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) were evaluated in six laboratories of university hospitals. Twenty serum samples were obtained from 12 neutropenic patients including 6 with invasive aspergillosis. These samples were blinded and sent to each center together with eight blinded ELISA-negative serum samples spiked with known concentrations of galactomannan. The centers were provided with ELISA microtiter plates from a single batch and a detailed protocol. Ten clinical samples showed ELISA reactivity, while 10 samples were ELISA negative. The mean coefficient of variation (CV) of the optical density values was 4.24% within a single assay and 25.6% between runs. The interassay CV of the ratios for the serum samples tested was 18.6%. Analysis of ordinal interpretation of the ELISA result (i.e., negative, gray zone, or positive) showed excellent reproducibility. Recalculation of the cutoff values for positive and negative samples suggested that the cutoff level recommended by the manufacturer could be lowered from 1.0 to 0.8 for negative samples and from 1.5 to 1.0 for positive samples. The intra- and interlaboratory reproducibilities were excellent when the ELISA results were interpreted as ordinal data, but considerable variation in optical density values and, to a lesser extent, in the ratios for the serum samples tested, was observed between runs. High assay variability was also found for serum samples spiked with known concentrations of galactomannan. Therefore, antigen titers in serum samples from a single patient, measured in different runs, should be compared with caution.