bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Qualitative-Quantitative Nested PCR for Detection of JC Virus in Cerebrospinal Fluid: High Potential for Evaluation and Monitoring of Progressive Multifocal Leukoencephalopathy in AIDS Patients Receiving Highly Active Antiretroviral Therapy
Authors: García de Viedma Darío , Alonso Roberto , Miralles Pilar , Berenguer Juan , Rodriguez-Créixems Marta , Bouza Emilio
Published: December 2019

JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a central nervous system infection that mainly affects AIDS patients. The extensive application of highly active antiretroviral therapy (HAART) is leading to the appearance of “long-term” survival PML patients. A reliable and feasible qualitative-quantitative test for both the detection of JCV and follow-up of its viral burden in this emerging group of patients is clearly required. With this aim, a dual qualitative-quantitative nested PCR is presented in this study for the analysis of JCV DNA in cerebrospinal fluid (CSF). Two newly designed internal controls, one competitive and the other noncompetitive, have been constructed to adapt this PCR to either measure the JCV burden or to allow a highly confident determination of JCV presence or clearance. The analytical sensitivity of the technique allows the detection of 0.01 fg (three genomes) of JCV DNA. Its qualitative application has been evaluated by analyzing single CSF samples from a group of 17 patients with PML and a control group of 20 patients with diverse neurological conditions other than PML, yielding sensitivity and specificity values of 100 and 90%, respectively. The quantitative application has been evaluated in vitro in blind tests with samples including serial dilutions of JCV, and in all cases the samples were successfully ordered considering the JCV titer. The dual quantitative-qualitative application offered by this nested PCR may provide an answer to the new requirements for evaluating and finely monitoring PML in AIDS patients receiving HAART.
Spread of Amikacin Resistance in
Acinetobacter baumannii
Strains Isolated in Spain Due to an Epidemic Strain
Authors: Vila Jordi , Ruiz Joaquim , Navia Margarita , Becerril Berta , Garcia Isabel , Perea Sofia , Lopez-Hernandez Inmaculada , Alamo Isabel , Ballester Frederic , Planes Anna M. , Martinez-Beltran Jesus , De Anta Teresa Jimenez
Published: December 2019

Sixteen amikacin-resistant clinical
Acinetobacter baumannii
isolates from nine different hospitals in Spain were investigated to determine whether the high incidence of amikacin-resistant
A. baumannii
was due to the dissemination of an amikacin-resistant strain or to the spread of an amikacin resistance gene. The epidemiological relationship studied by repetitive extragenic palindromic PCR and low-frequency restriction analysis of chromosomal DNA showed that the same clone was isolated in eight of nine hospitals, although other clones were also found. The strains were studied for the presence of the
aph(3′)-VIa
and
aac(6′)-I
genes, which encode enzymes which inactivate amikacin, by PCR. All 16 clinical isolates had positive PCRs with primers specific for the amplification of the
aph(3′)-VIa
gene, whereas none had a positive reaction for the amplification of the
aac(6′)-I
gene. Therefore, the high incidence of amikacin resistance among clinical
A. baumannii
isolates in Spain was mainly due to an epidemic strain, although the spread of the
aph(3′)-VI
gene cannot be ruled out.
Assessment of Use of the COBAS AMPLICOR System with BACTEC 12B Cultures for Rapid Detection of Frequently Identified Mycobacteria
Authors: Ninet B. , Rohner P. , Metral C. , Auckenthaler R.
Published: December 2019

The use of the COBAS AMPLICOR System (Roche Molecular Diagnostics, Basel, Switzerland), the only automated system for PCR testing, was evaluated for a rapid identification of mycobacteria with positive BACTEC 12B cultures. Two hundred ninety-six specimens with a growth index of ≥30 were analyzed for the presence of
Mycobacterium tuberculosis
complex,
Mycobacterium avium
, and
Mycobacterium intracellulare
. Compared to traditional methods and provided that samples with PCR inhibition are retested at a 1:10 dilution, the sensitivity and specificity of the COBAS AMPLICOR System with BACTEC 12B cultures were 100 and 98%, respectively. The COBAS AMPLICOR method is rapid and reliable for identifying the most common mycobacteria in cultures.
Human Phaeohyphomycotic Osteomyelitis Caused by the Coelomycete
Phomopsis
Saccardo 1905: Criteria for Identification, Case History, and Therapy
Authors: Sutton Deanna A. , Timm William D. , Morgan-Jones Gareth , Rinaldi Michael G.
Published: December 2019

The Sphaeropsidales, coelomycetous fungi producing asexual conidia within enclosed conidiomata (pycnidia), are saprobic on numerous vascular plants. Despite their ubiquitous nature, only a limited number of genera have been documented as causing human disease. We report what we believe to be the first human case of osteomyelitis due to a
Phomopsis
species in a chronically immunosuppressed female. The patient developed a subcutaneous abscess on the distal phalanx of the right fourth finger complicated by osteomyelitis. Operative specimens revealed fungal hyphae and a pure culture of mould. The patient was treated with a 6-month course of itraconazole. At 16 months of follow-up, she remained free of recurrence.
Phomopsis
species differ from the similar, more frequently reported
Phoma
species by having immersed, thick-walled, multiloculate conidiomata and by the production of alpha (short, ellipsoidal) and beta (long, filamentous) conidia.
Comparison of Simple and Rapid Methods for Identifying Enterococci Intrinsically Resistant to Vancomycin
Authors: Hanson Kevan L. , Cartwright Charles P.
Published: December 2019

Three different methodologies, reduction of litmus milk (LM) and acidification of arabinose (ARA), acidification of methyl-α-
d
-glucopyranoside (MGP), and rapid motility (RM), for differentiating isolates of
Enterococcus casseliflavus
and
Enterococcus gallinarum
(intrinsically vancomycin-resistant enterococci [IVRE]) from
Enterococcus faecalis
and
Enterococcus faecium
were evaluated. All 33 isolates of
E. faecalis
tested reduced LM within 4 h and were negative in all other tests, while the 53 isolates of
E. faecium
were ARA positive only. In contrast, 45 of 46 (98%) IVRE isolates examined (26
E. casseliflavus
and 20
E. gallinarum
isolates) acidified MGP, 41 of 46 (89%) were LM and ARA positive, and 45 of 46 (98%) were RM positive. Acidification of MGP was therefore the single most useful test for differentiating IVRE from vancomycin-resistant
E. faecium
and
E. faecalis
; however, a combination of LM-ARA and RM testing enabled the correct designation of organisms without the need for overnight incubation.
Comparison of the API Candida System with the AUXACOLOR System for Identification of Common Yeast Pathogens
Authors: Campbell Colin K. , Davey Kate G. , Holmes Ann D. , Szekely Adrien , Warnock David W.
Published: December 2019

Two commercial systems for the identification of yeasts were evaluated by using 159 clinical isolates that had also been identified by conventional biochemical and morphological methods. The API Candida system correctly identified 146 isolates (91.8%), and the AUXACOLOR system correctly identified 145 isolates (91.2%). However, of the 146 isolates identified by the API Candida system, 23 required supplemental biochemical tests or morphological assessment to obtain the correct identification. The AUXACOLOR system gave no identification in 13 cases (8.2%), while the API Candida system gave an unreadable profile in only one case. Incorrect identifications were more common with the API Candida system (12 isolates; 7.5%) than with the AUXACOLOR system (1 isolate; 0.6%).
Chronic Urinary Tract Infection Due to
Candida utilis
Authors: Hazen Kevin C. , Theisz Gordon W. , Howell Susan A.
Published: December 2019

An elderly male was seen at an outpatient urology clinic over a period of 3 years with repeat urine specimens containing 10
4
to 10
5
CFU of a “
Candida
species, not
C. albicans
.” The urine specimens were described as infected due to the presence of pyuria, but no antifungal therapy was administered. On two occasions, the patient presented to the emergency room and urine specimens were sent to the clinical microbiology laboratory. On both occasions, a yeast was isolated at concentrations of >10
5
CFU/ml. The organism was identified as the anamorphic yeast
Candida utilis
(teleomorph:
Pichia jadinii
) by conventional methods. Molecular methods, including karyotyping and restriction enzyme analysis, confirmed that the isolates were identical and were
C. utilis
. The patient developed benign prostatic hypertrophy and chronic obstructive pulmonary disease during the 3-year course. This report is the first demonstration of the isolation of the industrially important yeast
C. utilis
from a urinary tract infection. In the present case, the organism was associated with chronic, symptomatic disease. The significance of this unusual, low-virulence isolate from a case of urinary tract infection is discussed.
Cryptococcus neoformans
var.
grubii
: Separate Varietal Status for
Cryptococcus neoformans
Serotype A Isolates
Authors: Franzot Sarah P. , Salkin Ira F. , Casadevall Arturo
Published: December 2019

Cryptococcus neoformans
var.
neoformans
presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the
URA5
sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting
C. neoformans
var.
neoformans
to isolates which are serotype D and describing a new variety,
C. neoformans
var.
grubii
, for serotype A isolates.
Aerobic and Anaerobic Microbiology of Surgical-Site Infection Following Spinal Fusion
Authors: Brook Itzhak , Frazier Edith H.
Published: December 2019

The aerobic and anaerobic microbiology of surgical-site infections (SSI) following spinal fusion was retrospectively studied. This was done by reviewing the clinical and microbiological records at the Naval Hospital in Bethesda, Md., from 1980 to 1992. Aspirates of pus from 25 infection sites showed bacterial growth. Aerobic bacteria only were recovered from 9 (36%) specimens, anaerobic bacteria only were recovered from 4 (16%), and mixed aerobic and anaerobic bacteria were recovered from 12 (48%). Sixty isolates were recovered: 38 aerobes (1.5 isolates per specimen) and 22 anaerobes (0.9 isolate per specimen). The predominant aerobes were
Escherichia coli
(
n
= 8) and
Proteus
sp. (
n
= 7). The predominant anaerobes were
Bacteroides fragilis
group (
n
= 9) and
Peptostreptococcus
sp. (
n
= 6) isolates. An increase in recovery of
E. coli
and
B. fragilis
was noted in patients with bowel or bladder incontinence. This study highlights the polymicrobial nature of SSI and the importance of anaerobic bacteria in SSI following spinal fusion.
Use of Restriction Fragment Analysis and Sequencing of a Serotype-Specific Region To Type Adenovirus Isolates
Authors: Li Quan-Gen , Henningsson Anna , Juto Per , Elgh Fredrik , Wadell Göran
Published: December 2019

Restriction fragment analysis and sequencing of a serotype-specific region were used to type 12 and 2 clinical isolates, respectively. Both molecular methods produced clear-cut results that completely correlated with that of the neutralization test.
Identification of
Shigella flexneri
Subserotype 1c in Rural Egypt
Authors: El-Gendy Atef , El-Ghorab Nemat , Lane Edward M. , Elyazeed Remon Abu , Carlin Nils I. A. , Mitry Mary M. , Kay Bradford A. , Savarino Stephen J. , Peruski Leonard F.
Published: December 2019

In a population-based study of diarrhea in rural, northern Egypt, 60
Shigella flexneri
strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional
S. flexneri
subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that
S. flexneri
subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.
Comparison of Isolation Media for Recovery of
Burkholderia cepacia
Complex from Respiratory Secretions of Patients with Cystic Fibrosis
Authors: Henry Deborah , Campbell Maureen , McGimpsey Colleen , Clarke Alison , Louden Laurie , Burns Jane L. , Roe Martha H. , Vandamme Peter , Speert David
Published: December 2019

Burkholderia cepacia
selective agar (BCSA) has previously been devised for isolation of
B. cepacia
from respiratory secretions of patients with cystic fibrosis and tested under research laboratory conditions. Here we describe a study in which BCSA, oxidation-fermentation polymyxin bacitracin lactose agar (OFPBL), and
Pseudomonas cepacia
agar (PCA) were compared in routine culture procedures for the ability to grow
B. cepacia
and inhibit other organisms. Three hundred twenty-eight specimens from 209 patients at two pediatric centers and 328 specimens from 109 adults were tested. Plates were inoculated, incubated, and read for quality and quantity of growth at 24, 48, and 72 h. Five (1.5%) specimens from 4 (1.9%) children and 75 (22.9%) specimens from 16 (14.7%) adults grew
B. cepacia
complex. At 24, 48, and 72 h, BCSA achieved 43, 93, and 100% detection, respectively; OFPBL achieved 26, 84, and 96%, respectively; and PCA achieved 33, 74, and 84% detection, respectively. Quality was assessed as pinpoint or good growth. At 24 h, most cultures growing
B. cepacia
complex had pinpoint colonies. By 48 and 72 h, 48 and 69% of
B. cepacia
complex cultures, respectively, had good growth on BCSA, while on OFPBL 19 and 30%, respectively, had good growth and on PCA 11 and 18%, respectively, had good growth. BCSA was superior to OFPBL and PCA in suppressing organisms other than
B. cepacia
complex; 40 non-
B. cepacia
complex organisms were isolated from BCSA, 263 were isolated from OFPBL, and 116 were isolated from PCA. We conclude that BCSA is superior to OFPBL and PCA in its ability to support the growth of
B. cepacia
complex and to suppress other respiratory organisms.
Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture ofMycobacterium aviumsubsp.paratuberculosisfrom Sheep
Authors: Whittington R. J. , Marsh I. , McAllister S. , Turner M. J. , Marshall D. J. , Fraser C. A.
Published: December 2019

Definitive diagnosis of Johne’s disease in ruminants depends on confirming the presence of the causative bacterium,Mycobacterium aviumsubsp.paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture ofM. aviumsubsp.paratuberculosisfrom sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture ofM. aviumsubsp.paratuberculosisfrom sheep with Johne’s disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne’s disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the “gold standard” test for detection of ovine Johne’s disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains ofM. aviumsubsp.paratuberculosis. The sensitivity of detection ofM. aviumsubsp.paratuberculosison solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains ofM. aviumsubsp.paratuberculosisin both liquid and solid media.
Human Immunodeficiency Virus Antibody Testing by Enzyme-Linked Fluorescent and Western Blot Assays Using Serum, Gingival-Crevicular Transudate, and Urine Samples
Authors: Martínez Prudencio Martínez , Torres Antonio Rodríguez , Ortiz de Lejarazu Raul , Montoya Ana , Martín José Francisco , Eiros José María
Published: December 2019

The aim of the present study was to evaluate the possible utilization of saliva and urine as alternative samples to serum for the diagnosis of human immunodeficiency virus (HIV) infection. A total of 302 individuals participated in the study: 187 HIV-infected individuals (106 had Centers for Disease Control and Prevention [CDC] stage II infection, 19 had CDC stage III infection, and 62 had CDC stage IV infection) and 115 noninfected persons (46 of the noninfected persons were blood donors and 69 belonged to a group at high risk of HIV infection). Paired saliva and urine samples were taken from each of the participants in the study. The presence of HIV-specific antibodies was detected by an enzyme-linked fluorescent assay (ELFA), and the result was confirmed by Western blot analysis (WB). The ELFA with saliva gave maximum sensitivity and specificity values, while ELFA had lower sensitivity (95.2%) and specificity (97.4%) values for detection of HIV antibody in urine samples. WB with all saliva samples fulfilled the World Health Organization criterion for positivity, while only 96.8% of the urine samples were confirmed to be positive by WB. Among the four reactivity patterns found by WB of these alternative samples, the most frequent included bands against three groups of HIV structural proteins (was ENV, POL, and GAG). The reactivity bands most frequently observed were those for the proteins gp160 and gp120. The least common reactivity band was the band for protein p17. The detection of HIV antibodies in saliva samples by means of ELFA with the possibility of later confirmation by WB makes saliva an alternative to serum for possible use in the diagnosis of infection. In contrast, HIV antibody detection in urine samples by the same methodology (ELFA) could be taken into consideration for use in epidemiological studies.
Detection of Epstein-Barr Virus DNA in Sera from Transplant Recipients with Lymphoproliferative Disorders
Authors: Limaye Ajit P. , Huang Meei-Li , Atienza Ederlyn E. , Ferrenberg James M. , Corey Lawrence
Published: December 2019

Early diagnosis of Epstein-Barr Virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is important because many patients respond to reduction in immunosuppression, especially if PTLD is detected at an early stage. Previous studies have found elevated EBV DNA levels in blood from patients with PTLD, but these assays required isolation of cellular blood fractions and quantitation. We evaluated the presence of cell-free EBV DNA in serum from solid-organ transplant recipients as a marker for PTLD. Five of 6 transplant recipients with histopathologically documented PTLD had EBV DNA detected in serum at the time of diagnosis (sensitivity = 83%), compared with 0 of 16 matched transplant recipients without PTLD (specificity = 100%) (
P
< 0.001 [Fisher’s exact test]). Furthermore, EBV DNA was detected in serum 8 and 52 months prior to the diagnosis of PTLD in two of three patients for whom stored sera were analyzed. Detection of EBV DNA in serum appears to be a useful marker for the early detection of PTLD in solid-organ transplant recipients. Further studies to define the role of such assays in evaluating solid-organ transplant patients at risk for PTLD are warranted.
Bartonella koehlerae
sp. nov., Isolated from Cats
Authors: Droz Sara , Chi Banghee , Horn Elke , Steigerwalt Arnold G. , Whitney Anne M. , Brenner Don J.
Published: December 2019

Two of the 25
Bartonella
isolates recovered during a prevalence study of
Bartonella henselae
bacteremia in domestic cats from the greater San Francisco Bay region were found to differ phenotypically and genotypically from all prior
B. henselae
isolates. These isolates, C-29 and C-30, which were recovered from the blood of two pet cats belonging to the same household, grew on chocolate agar as pinpoint colonies following 14 days of incubation at 35°C in a candle jar but failed to grow on heart infusion agar supplemented with 5% rabbit blood. Additional phenotypic characteristics distinguished the isolates C-29 and C-30 from other feline
B. henselae
isolates. The restriction patterns obtained for C-29 and C-30 by citrate synthase PCR-restriction fragment length polymorphism (RFLP) analysis as well as by genomic RFLP could not be distinguished from each other but were distinctly different from that of the
B. henselae
type strain. In reciprocal reactions, DNAs from strains C-29 and C-30 were 97 to 100% related under optimal and stringent DNA reassociation conditions, with 0 to 0.5% divergence within related sequences. Labeled DNA from the type strain of
B. henselae
was 61 to 65% related to unlabeled DNAs from strains C-29 and C-30 in 55°C reactions, with 5.0 to 5.5% divergence within the related sequences, and 31 to 41% related in stringent, 70°C reactions. In reciprocal reactions, labeled DNAs from strains C-29 and C-30 were 68 to 92% related to those of the
B. henselae
type strain and other
B. henselae
strains, with 5 to 7% divergence. The 16S rRNA gene sequence of strain C-29 was 99.54% homologous to that of the type strain of
B. henselae
. On the basis of these findings, the two isolates C-29 and C-30 are designated a new species of
Bartonella
, for which we propose the name
Bartonella koehlerae
. The type strain of
Bartonella koehlerae
is strain C-29 (ATCC 700693).
Facklamia languida
sp. nov., Isolated from Human Clinical Specimens
Authors: Lawson Paul A. , Collins Matthew D. , Falsen Enevold , Sjöden Berit , Facklam Richard R.
Published: December 2019

Three strains of a gram-positive catalase-negative, facultatively anaerobic coccus-shaped organism originating from human clinical samples were characterized by phenotypic and molecular taxonomic methods. Sequencing of genes encoding 16S rRNA showed that the strains are phylogenetically closely related (99.9 to 100% sequence similarity) and represent a new subline within the genus
Facklamia
. The unknown bacterium was readily distinguished from all currently described species of the genus
Facklamia
(viz.,
Facklamia hominis
,
Facklamia ignava
, and
Facklamia sourekii
) by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as
Facklamia languida
sp. nov. The type strain of
F. languida
is CCUG 37842.
Rapid Identification of
Staphylococcus aureus
by Using Fluorescent Staphylocoagulase Assays
Authors: Holliday M. G. , Ford M. , Perry J. D. , Gould F. K.
Published: December 2019

Two rapid (1-h) assays for the detection of
Staphylococcus aureus
staphylocoagulase were developed by using the fluorogenic thrombin substrates
N-t
-boc-Val-Pro-Arg-7-amido-4-methylcoumarin (VPA) and
N-t
-boc-β-benzyl-Asp-Pro-Arg-7-amido-4-methylocoumarin (BB). The assays were compared to the tube coagulase test and latex agglutination (LA) (Sanofi Diagnostics Pasteur, Guildford, Surrey, United Kingdom) by using 406 clinical isolates of staphylococci, and they produced positive and negative predictive values of 99.2 and 99.1% for LA, 98.9 and 92.7% for VPA, and 98.9 and 99.1% for BB. Fluorescent assays used colonies from solid media, thereby eliminating the need for broth cultures, and were performed in microtiter trays, thus making them suitable for large-scale screening.
Comparison of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma as Measured by the NucliSens Nucleic Acid Sequence-Based Amplification and Quantiplex Branched-DNA Assays
Authors: Ginocchio C. C. , Tetali S. , Washburn D. , Zhang F. , Kaplan M. H.
Published: December 2019

This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (
P
< 0.0001), although the Quantiplex and NucliSens values were significantly different (
P
< 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays:
r
= 0.912,
P
< 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.
Serum Is More Suitable than Whole Blood for Diagnosis of Systemic Candidiasis by Nested PCR
Authors: Bougnoux M.-E. , Dupont C. , Mateo J. , Saulnier P. , Faivre V. , Payen D. , Nicolas-Chanoine M.-H.
Published: December 2019

PCR assays for the diagnosis of systemic candidiasis can be performed either on serum or on whole blood, but results obtained with the two kinds of samples have never been formally compared. Thus we designed a nested PCR assay in which five specific inner pairs of primers were used to amplify specific targets on the rRNA genes of
Candida albicans
,
C. tropicalis
,
C. parapsilosis
,
C. krusei
, and
C. glabrata
. In vitro, the lower limit of detection of each nested PCR assay was 1 fg of purified DNA from the corresponding
Candida
species. In rabbits with candidemia of 120 minutes’ duration following intravenous (i.v.) injection of 10
8
CFU of
C. albicans
, the sensitivities of the PCR in serum and whole blood were not significantly different (93 versus 86%). In other rabbits, injected with only 10
5
CFU of
C. albicans
, detection of candidemia by culture was possible for only 1 min, whereas DNA could be detected by PCR in whole blood and in serum for 15 and 150 min, respectively. PCR was more often positive in serum than in whole blood in 40 culture-negative samples (27 versus 7%;
P
< 0.05%). Lastly, experiments with rabbits injected i.v. with 20 or 200 μg of purified
C. albicans
DNA showed that PCRs were positive in serum from 30 to at least 120 min after injection, suggesting that the clearance of free DNA is slow. These results suggest that serum is the sample of choice, which should be used preferentially over whole blood for the diagnosis of systemic candidiasis by PCR.
Amplification of the Six Major Human Herpesviruses from Cerebrospinal Fluid by a Single PCR
Authors: Minjolle Sophie , Michelet C. , Jusselin I. , Joannes M. , Cartier F. , Colimon R.
Published: December 2019

We used a novel type of primer system, a system that uses stair primers, in which the primer sequences are based on consensus sequences in the DNA polymerase gene of herpesvirus to detect herpesviruses by PCR. A single PCR in a single tube detected the six major herpesviruses that infect the central nervous system: herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6). We used the technique to analyze 142 cerebrospinal fluid (CSF) samples that had been stored at −80°C and compared the results with those obtained previously for the same samples by standard, targeted PCR. Four hundred one targeted PCR tests had been run with the 142 samples to detect HSV-1, HSV-2, CMV, and VZV; screening for EBV and HHV-6 was not prescribed when the samples were initially taken. Eighteen CSF samples tested positive by classic targeted PCR. The herpesvirus consensus PCR detected herpesviruses in 37 samples, including 3 samples with coinfections and 17 viral isolates which were not targeted. Two samples identified as infected by the targeted PCR tested negative by the consensus PCR, and eight samples that tested positive by the consensus PCR were negative by the targeted PCR. One hundred three samples scored negative by both the targeted and the consensus PCRs. This preliminary study demonstrates the value of testing for six different herpesviruses simultaneously by a sensitive and straightforward technique rather than screening only for those viruses that are causing infections as suggested by clinical signs.
Multicenter Comparison of the Digene Hybrid Capture CMV DNA Assay (Version 2.0), the pp65 Antigenemia Assay, and Cell Culture for Detection of Cytomegalovirus Viremia
Authors: Mazzulli Tony , Drew Lawrence W. , Yen-Lieberman Belinda , Jekic-McMullen Dragana , Kohn Debra J. , Isada Carlos , Moussa George , Chua Robert , Walmsley Sharon
Published: December 2019

We compared the Digene Hybrid Capture CMV DNA Assay version 2.0, the pp65 antigenemia assay, traditional tube culture, and shell vial culture for the detection of cytomegalovirus (CMV) viremia in several patient populations at three centers. Of 561 blood specimens collected from 402 patients, complete clinical and laboratory data were available for 489. Using consensus definitions for true positives and true negatives, the sensitivities of the Hybrid Capture assay, antigenemia, shell vial, and tube culture were 95, 94, 43, and 46%, respectively. The specificities of the Hybrid Capture assay and antigenemia were 95 and 94%, respectively. At all three study sites, the detected level of CMV viremia was significantly higher with the Hybrid Capture assay or antigenemia than with shell vial and tube culture. In a group of 131 healthy nonimmunosuppressed volunteers, the Hybrid Capture assay demonstrated a specificity of over 99%. The Hybrid Capture assay is a standardized assay that is simple to perform and can utilize whole blood specimens that have been stored for up to 48 h. The high sensitivity and specificity of the Hybrid Capture assay along with its simplicity and flexibility make it a clinically useful assay for the detection of CMV viremia in immunocompromised or immunosuppressed patients. Further evaluation to determine its role in predicting CMV disease and for monitoring the therapeutic response to anti-CMV therapy is needed.
Detection of
Bartonella henselae
DNA by Two Different PCR Assays and Determination of the Genotypes of Strains Involved in Histologically Defined Cat Scratch Disease
Authors: Sander Anna , Posselt Miriam , Böhm Norbert , Ruess Michael , Altwegg Martin
Published: December 2019

Cat scratch disease (CSD) is a common cause of subacute regional lymphadenopathy, not only in children but also in adults. Serological and molecular studies demonstrated that
Bartonella henselae
is the etiologic agent in most cases of CSD. Amplification of
B. henselae
DNA in affected tissue and detection of antibodies to
B. henselae
are the two mainstays in the laboratory diagnosis of CSD. We designed a retrospective study and investigated formalin-fixed, paraffin-embedded lymph nodes from 60 patients (25 female, 35 male) with histologically suspected CSD by PCR amplification. The sensitivities of two different PCR assays were compared. The first primer pair amplified a 296-bp fragment of the 16S rRNA gene in 36 of the 60 samples, corresponding to a sensitivity of 60%. The second primer pair amplified a 414-bp fragment of the
htrA
gene in 26 of the 60 lymph nodes, corresponding to a sensitivity of 43.3%.
Bartonella
DNA could be detected in a total of 39 (65%) of the 60 lymph nodes investigated. However, histopathologic findings are typical but not specific for CSD and cannot be considered as a “gold standard” for diagnosis of CSD. The sensitivity of the PCR assays increased from 65 to 87% if two criteria (histology and serology) were used in combination for diagnosis of CSD. Two genotypes (I and II) of
B. henselae
are described as being involved in CSD. Genotype I was found in 23 (59%) and genotype II was found in 9 (23%) of the 39 PCR-positive lymph nodes. Seven (18%) lymph nodes were negative in both type-specific PCR assays. Thirty (50%) of our 60 patients were younger than 20 years old (15 were younger than 10 years), 20 (33%) were between 21 and 40 years old, and 10 (17%) patients were between 41 and 84 years old. Our data suggest that detection of
Bartonella
DNA in patients’ samples might confirm the histologically suspected diagnosis of CSD.
Studies of Genetic Relationships between Bovine, Caprine, Cervine, and Rangiferine Alphaherpesviruses and Improved Molecular Methods for Virus Detection and Identification
Authors: Ros Carlos , Belák Sándor
Published: December 2019

The glycoprotein B (gB) and D (gD) genes from five ruminant alphaherpesviruses, bovine herpesvirus 1 (BHV-1), bovine herpesvirus 5 (BHV-5), caprine herpesvirus 1 (CapHV-1), cervine herpesvirus 1, and rangiferine herpesvirus 1, were partially sequenced. The nucleotide sequence alignments revealed a highly conserved gB gene, with homologies ranging between 87.2 and 99.6%, and a more variable gD gene, with homologies ranging between 71.3 and 98.9%. The phylogenetic analysis of the gB and gD nucleotide and deduced amino acid sequences revealed that BHV-5 is the most closely related virus to the BHV-1 subtype 1 and BHV-1 subtype 2 cluster and that CapHV-1 is the most distantly related virus. The phylogenetic data showed a close relationship of all the studied viruses with suid herpesvirus 1. On the basis of sequence data for the gB gene, a nested PCR combined with restriction enzyme analysis (REA) of the PCR products was developed for the simultaneous detection and identification of the viruses that were studied. Nested primers from highly conserved sequence stretches were selected in order to amplify a region of 294 bp in all five viruses, and a subsequent REA of the PCR products allowed specific identification. A mimic molecule that served as an internal standard of the amplification efficiency was constructed. The practical diagnostic applicability of the assay was evaluated with clinical samples consisting of semen and organ specimens from experimentally infected animals.
Phenotypic and Genomic Variation among
Staphylococcus epidermidis
Strains Infecting Joint Prostheses
Authors: Galdbart Jacques-Olivier , Morvan Anne , Desplaces Nicole , el Solh Nevine
Published: December 2019

We studied the
Sma
I and
Sst
II macrorestriction patterns of 54
Staphylococcus epidermidis
strains isolated from 14 patients infected following the implantation of joint prostheses. Multiple strains from pus and infected tissue specimens of each patient were selected on the basis of different colony morphologies and drug resistance patterns. The same criteria were used to select 23
S. epidermidis
strains from hand swabs of eight healthy individuals. For 10 of the 14 patients, all the intrapatient strains appeared to be closely or possibly related, whereas related strains were detected in the skin flora of only one of the eight healthy individuals. This observation suggests that, in most cases, the patients were infected by a single
S. epidermidis
clone which subsequently underwent rearrangements that yielded derivatives with divergent phenotypes and, occasionally, divergent macrorestriction patterns. The four patients whose specimens contained unrelated
S. epidermidis
strains were probably infected with several polyclonal strains.
Molecular Characterization of a New Ribotype of
Vibrio cholerae
O139 Bengal Associated with an Outbreak of Cholera in Bangladesh
Authors: Faruque Shah M. , Siddique A. K. , Saha Manujendra N. , Asadulghani  , Rahman M. Mostafizur , Zaman K. , Albert M. John , Sack David A. , Sack R. Bradley
Published: December 2019

Vibrio cholerae
O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of
V. cholerae
O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by
V. cholerae
O139 led us to analyze strains collected from the outbreak and compare them with
V. cholerae
O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated
V. cholerae
O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic
V. cholerae
O139. All strains carried the genes for toxin-coregulated pili (
tcpA
and
tcpI
) and accessory colonization factor (
acfB
), the regulatory gene
toxR
, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXΦ) and belonged to a previously described
ctxA
genotype. Comparative analysis of the
rfb
gene cluster by PCR revealed the absence of a large region of the O1-specific
rfb
operon downstream of the
rfaD
gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of
V. cholerae
O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus,
V. cholerae
O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.
Evaluation of the INNO-LIA HTLV I/II Assay for Confirmation of Human T-Cell Leukemia Virus-Reactive Sera in Blood Bank Donations
Authors: Sabino Ester C. , Zrein Maan , Taborda Carlos P. , Otani Marcia M. , Ribeiro-Dos-Santos Gabriela , Sáez-Alquézar Amadeo
Published: December 2019

We have evaluated a new serological confirmatory test (INNO-LIA HTLV I/II Ab [INNO-LIA]) for human T-cell leukemia virus (HTLV) using a large collection of samples from Brazilian blood donors (São Paulo region) and compared the results with those obtained by Western blotting (WB) tests (WB2.3 and WB2.4). Blood donations were initially screened by enzyme-linked immunosorbent assays (ELISAs) based on viral lysates, and repeatedly reactive samples were further tested by WB2.3. When available, samples were also tested by PCR, two additional ELISAs based on recombinant antigens (recombinant ELISAs), a new-generation WB assay (WB2.4), and the INNO-LIA. Of the 18,169 samples tested, 292 (1.61%) were repeatedly reactive in the ELISAs (viral lysate based) and were further tested by WB2.3; 97 were positive (19 that were typed as HTLV type I [HTLV-I], 12 that were typed as HTLV type II [HTLV-II], and 66 that were nontypeable), 17 were negative, and 178 had indeterminate results. Of the samples with indeterminate results, 172 were tested by INNO-LIA, which could resolve 153 samples as negative. Regarding the positive samples, WB2.3 and INNO-LIA produced concordant results for all HTLV-I-positive samples, whereas for HTLV-II they agreed for 10 of 12 samples; the 2 samples with discordant results were considered to be positive for HTLV-II by WB with WB2.3 but negative for HTLV-II by INNO-LIA and the two recombinant ELISAs. Furthermore, of the 66 nontypeable samples, 60 underwent testing by INNO-LIA; 54 turned out to be negative by the latter test as well as by recombinant ELISAs. In conclusion, the new serological confirmatory assay for HTLV (INNO-LIA HTLV I/II Ab) resolved the results for the majority of the indeterminate and positive-untypeable samples frequently observed by WB assays.
Prevalence of
Borrelia burgdorferi
in
Ixodes ricinus
Ticks in Urban Recreational Areas of Helsinki
Authors: Junttila Juha , Peltomaa Miikka , Soini Hanna , Marjamäki Merja , Viljanen Matti K.
Published: December 2019

Lyme borreliosis, an infection caused by the tick-borne spirochete
Borrelia burgdorferi
, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of
B. burgdorferi
sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688
Ixodes ricinus
ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis. The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with
B. burgdorferi
sensu lato varied from 19 to 55%, with the average being 32%.
Borellia afzelii
was the most predominant genospecies in all the areas, and no
B. burgdorferi
sensu stricto isolates were detected. Only two ticks were concurrently infected with both
B. afzelii
and
Borrelia garinii
. Dark-field microscopy gave more positive results for
B. burgdorferi
than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.
Evaluation of Human Seroreactivity to
Bartonella
Species in Sweden
Authors: Holmberg M. , McGill S. , Ehrenborg C. , Wesslén L. , Hjelm E. , Darelid J. , Blad L. , Engstrand L. , Regnery R. , Friman G.
Published: December 2019

Among the species that compose the expanding genus
Bartonella
, thus far only
B. henselae
and
B. quintana
have reportedly been isolated from humans in Europe. To evaluate the prevalence of
Bartonella
infection in Sweden, we conducted a retrospective serological examination of 126 human serum samples. These samples were analyzed for antibodies to
B. henselae
,
B. quintana
, and
B. elizabethae
. Serum samples from 100 blood donors, who spanned the ages of 20 to 60 and had no apparent clinical signs of illness, were also studied as a control group. An immunoglobulin G indirect fluorescence antibody assay revealed 4 and 8.3%
Bartonella
positivity rates for the blood donor and patient group, respectively, when a cutoff titer of ≥64 was chosen. Among the blood donors, four were seropositive to
B. elizabethae
; one of these also had concordant positive titer to
B. henselae
. In the patient group, 14 serum samples were positive against
Bartonella
spp. These serum specimens represented nine patients. In three of these seropositive patients, paired serum samples displayed a fourfold increase in antibody titer to at least one of the three antigens. These three patients are discussed. In this report we also present a case study of a 60-year-old Swedish male with fatal myocarditis. Postmortem serological analysis revealed a high titer against
B. elizabethae
. PCR and nucleotide sequencing of the myocardial tissue from this patient, and of liver tissue from one of the other three patients, showed sequences similar to
B. quintana
. The age, geographical origin, animal contacts, and serological response pattern to the different
Bartonella
antigens differed among the four patients. This study substantiates the presence of
Bartonella
spp. in Sweden, documents the seroreactivity to three
Bartonella
antigens in Swedish patients, and reports the first two cases of
B. quintana
-like infections in Sweden.
Comparison of E-Test and Broth Microdilution Methods for Antifungal Drug Susceptibility Testing of Molds
Authors: Szekely Adrien , Johnson Elizabeth M. , Warnock David W.
Published: December 2019

We compared the E test with a broth microdilution method, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 90 isolates of pathogenic molds (10
Absidia corymbifera
, 10
Aspergillus flavus
, 10
Aspergillus fumigatus
, 10
Aspergillus niger
, 10
Aspergillus terreus
, 10
Exophiala dermatitidis
, 10
Fusarium solani
, 10
Scedosporium apiospermum
, 5
Scedosporium prolificans
, and 5
Scopulariopsis brevicaulis
). Overall, there was 71% agreement between the results of the two methods for amphotericin B (E-test MICs within ±2 log
2
dilutions of broth microdilution MICs) and 88% agreement with the results for itraconazole. The overall levels of agreement (within ±2 log
2
dilutions) were ≥80% for 5 of the 10 species tested against amphotericin B and 8 of the 10 species tested against itraconazole. The best agreement between the results was seen with
A. fumigatus
and
A. terreus
(100% of results for both agents within ±2 log
2
dilutions). The poorest agreement was seen with
S. apiospermum
,
S. prolificans
, and
S. brevicaulis
tested against amphotericin B (20% of results within ±2 log
2
dilutions). In every instance, this low level of agreement was due to isolates for which the broth microdilution MICs were low but for which the E-test MICs were much higher. The E test appears to be a suitable alternative procedure for testing the susceptibility of
Aspergillus
spp. and some other molds to amphotericin B or itraconazole.