bluuebunny/crossref_metadata_2025 huggingface dataset:
SimulFluor Respiratory Screen for Rapid Detection of Multiple Respiratory Viruses in Clinical Specimens by Immunofluorescence Staining
Authors: Landry Marie L. , Ferguson David
Published: December 2019

A new rapid direct immunofluorescence assay (DFA) respiratory screen reagent for detection of seven common respiratory viruses (respiratory syncytial virus [RSV], influenza A and B viruses, parainfluenza virus types 1 to 3, and adenovirus) was compared with standard single or dual DFA reagents and culture. In total, 1,531 respiratory samples were adequate for testing with both SimulFluor Respiratory Screen (RS) reagent (Chemicon International, Temecula, Calif.) and single or dual DFA reagents. The RS DFA reagent detected 367 (98.4%) and single or dual DFA reagents detected 368 (98.7%) of 373 DFA-positive samples. In addition, the RS DFA reagent was equivalent to or better than culture for detection of all viruses except adenovirus. Only 15 of 799 (1.9%) RS-negative samples inoculated into cell cultures yielded respiratory virus isolates (one RSV, five influenza A virus, two influenza B virus, one parainfluenza virus, and six adenovirus). Sixty-six other virus isolates (13 rhinovirus, 24 cytomegalovirus, 28 herpes simplex virus type 1, and 1 enterovirus) were also recovered in culture. With cytospin preparation of slides, only 7.5% of samples submitted were deemed inadequate for DFA. The availability of a rapid DFA screening reagent for detection of multiple common respiratory viruses within 1 to 2 h of sample collection should be of great benefit in terms of patient management and infection control.
Pathogenic Clones versus Environmentally Driven Population Increase: Analysis of an Epidemic of the Human Fungal Pathogen
Coccidioides immitis
Authors: Fisher Matthew C. , Koenig Gina L. , White Thomas J. , Taylor John W.
Published: December 2019

For many pathogenic microbes that utilize mainly asexual modes of reproduction, it is unknown whether epidemics are due to either the emergence of pathogenic clones or environmentally determined increases in the population size of the organism. Descriptions of the genetic structures of epidemic populations, in conjunction with analyses of key environmental variables, are able to distinguish between these competing hypotheses. A major epidemic of coccidioidomycosis (etiologic agent,
Coccidioides immitis
) occurred between 1991 and 1994 in central California, representing an 11-fold increase above the mean number of cases reported from 1955 to 1990. Molecular analyses showed extensive genetic diversity, a lack of linkage disequilibria, and little phylogenetic structure, demonstrating that a newly pathogenic strain was not responsible for the observed epidemic. Epidemiological analyses showed that morbidity caused by
C. immitis
was best explained by the interaction between two variables, the lengths of droughts preceding epidemics and the amounts of rainfall. This shows that the principal factors governing this epidemic of
C. immitis
are environmental and not genetic. An important implication of this result is that the periodicity of cyclical environmental factors regulates the population size of
C. immitis
and is instrumental in determining the size of epidemics. This knowledge provides an important tool for predicting outbreaks of this pathogen, as well as a general framework that may be applied to determine the causes of epidemics of other fungal diseases.
Bacteremia Caused by a Strain of
Desulfovibrio
Related to the Provisionally Named
Desulfovibrio fairfieldensis
Authors: Loubinoux Julien , Mory Francine , Pereira Ines A. C. , Le Faou Alain E.
Published: December 2019

Eight isolates of
Desulfovibrio
spp. have been obtained over 5 years from abdominal or brain abscesses or blood. In seven patients these strains were part of a mixed flora. One strain was isolated in pure culture from the blood of a patient with peritonitis of appendicular origin. According to the 16S rRNA gene sequences, this strain was close to
Desulfovibrio fairfieldensis
. The present report describes the fourth isolate of this recently described species to be isolated in pure culture or as a predominant part of the flora and to be associated with infectious processes. Thus,
D. fairfieldensis
may possess a higher pathogenic potential than other
Desulfovibrio
species.
PCR Detection of Granulocytic Ehrlichiae in
Ixodes ricinus
Ticks and Wild Small Mammals in Western Switzerland
Authors: Liz Jorge S. , Anderes Laurence , Sumner John W. , Massung Robert F. , Gern Lise , Rutti Bernard , Brossard Michel
Published: December 2019

The presence of granulocytic ehrlichiae was demonstrated by PCR in
Ixodes ricinus
ticks and wild small mammals in Switzerland in two areas of endemicity for bovine ehrlichiosis. Six ticks (three females and three nymphs) (1.4%) of 417
I. ricinus
ticks collected by flagging vegetation contained ehrlichial DNA. A total of 201 small mammals from five species, wood mouse (
Apodemus sylvaticus
), yellow-necked mouse (
Apodemus flavicollis
), earth vole (
Pitymys subterraneus
), bank vole (
Clethrionomys glareolus
), and common shrew (
Sorex araneus
), were trapped. The analysis of
I. ricinus
mammals collected on 116 small mammals showed that nine
C. glareolus
voles and two
A. sylvaticus
mice hosted infected tick larvae. In these rodents, granulocytic ehrlichia infection was also detected in blood, spleen, liver, and ear samples. Further examinations of 190 small mammals without ticks or with noninfected ticks showed the presence of ehrlichial DNA in spleen and other tissues from six additional
C. glareolus
, three
A. flavicollis
, and one
S. araneus
mammals. This study suggests that
A. sylvaticus
,
A. flavicollis
,
S. araneus
, and particularly
C. glareolus
are likely to be natural reservoirs for granulocytic ehrlichiae. Partial 16S rRNA gene sequences of granulocytic ehrlichiae from ticks and rodents showed a high degree of homology (99 to 100%) with granulocytic ehrlichiae isolated from humans. In contrast,
groESL
heat shock operon sequence analysis showed a strong divergence (approximately 5%) between the sequences in samples derived from rodents and those derived from samples from questing ticks or from other published ehrlichia sequences. Dual infections with granulocytic ehrlichia and
Borrelia burgdorferi
were found in ticks and small mammals.
Imipenem Resistance of
Enterobacter aerogenes
Mediated by Outer Membrane Permeability
Authors: Bornet Charléric , Davin-Regli Anne , Bosi Claude , Pages Jean-Marie , Bollet Claude
Published: December 2019

Multidrug-resistant
Enterobacter aerogenes
strains are increasingly isolated in Europe and especially in France. Treatment leads to imipenem resistance, because of a lack of porin. We studied the evolution of resistance in 29 strains isolated from four patients during their clinical course. These strains belonged to the prevalent epidemiological type observed in France in previous studies (C. Bosi, et al., J. Clin. Microbiol. 37:2165–2169, 1999; A. Davin-Regli et al., J. Clin. Microbiol. 34:1474–1480, 1996). They also harbored a TEM-24 extended-spectrum β-lactamase-coding gene. Thirteen strains were susceptible to gentamicin and resistant to imipenem and cefepime. All of the patients showed
E. aerogenes
strains with this resistance after an imipenem treatment. One patient showed resistance to imipenem after a treatment with cefpirome. Twelve of these 13 strains showed a lack of porin. Cessation of treatment with imipenem for three patients was followed by reversion of susceptibility to this antibiotic and the reappearance of porins, except in one case. For one patient, we observed three times in the same day the coexistence of resistant strains lacking porin and susceptible strains possessing porin. The emergence of multidrug-resistant
E. aerogenes
strains is very disquieting. In our study, infection by
E. aerogenes
increased the severity of the patients' illnesses, causing a 100% fatality rate.
An Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Levels of the Dengue Virus Protein NS1 in the Sera of Infected Patients
Authors: Young Paul R. , Hilditch Paige A. , Bletchly Cheryl , Halloran Wendy
Published: December 2019

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 μg/ml, were found in acute-phase sera taken from some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.
Evaluation of the Indirect Hemagglutination Assay for Diagnosis of Acute Leptospirosis in Hawaii
Authors: Effler Paul V. , Domen Harry Y. , Bragg Sandra L. , Aye Tin , Sasaki David M.
Published: December 2019

Timely diagnosis of leptospirosis is important to ensure a favorable clinical outcome. The definitive serologic assay, the microscopic agglutination test (MAT), requires paired sera and is not useful for guiding early clinical management. The only screening test approved for use in the United States, the indirect hemagglutination assay (IHA), has not undergone extensive field evaluation. To assess the performance of the leptospirosis IHA in Hawaii, serum from patients evaluated for leptospirosis between 1992 and 1997 were tested with the IHA at the Hawaii State Laboratories Division and with the MAT at the Centers for Disease Control and Prevention. Leptospirosis was considered confirmed by a fourfold rise in MAT titer and/or a positive culture. A total of 92 (41%) of 226 specimens from 114 persons with confirmed leptospirosis were found positive by IHA. Only 18 (15%) of 119 specimens obtained within 14 days of onset were IHA positive, compared to 74 (69%) of 107 specimens collected more than 14 days after onset (
P
<0.001). Repeat testing ultimately resulted in 78 (68%) of the confirmed cases having at least one specimen found positive by IHA. Thirteen different presumptive infecting serogroups were identified among 251 specimens with an MAT titer of ≥200 and obtained from persons with confirmed or probable leptospirosis. Fifty (68%) of 73 specimens with Icterohaemorrhagiae as the presumptive infecting serogroup were found positive by IHA, compared to 44 (47%) of 93 specimens with Australis as the presumptive infecting serogroup (
P
, 0.01). The IHA test was positive for 3 (1%) of 236 specimens from 154 persons without leptospirosis. The sensitivity of the leptospirosis IHA in Hawaii was substantially below figures reported previously, particularly early in the course of illness, limiting its usefulness for diagnosing acute infection. Since the presumptive infecting serogroup affected IHA results and the prevalence of serovars varies with geography, the performance of the IHA should be assessed locally. More sensitive leptospirosis screening tests are needed in Hawaii.
Recombinant Antigens To DetectToxoplasma gondii-Specific Immunoglobulin G and Immunoglobulin M in Human Sera by Enzyme Immunoassay
Authors: Aubert D. , Maine G. T. , Villena I. , Hunt J. C. , Howard L. , Sheu M. , Brojanac S. , Chovan L. E. , Nowlan S. F. , Pinon J. M.
Published: December 2019

We have evaluated the diagnostic utility of elevenToxoplasma gondiirecombinant antigens (P22 [SAG2], P24 [GRA1], P25, P28 [GRA2], P29 [GRA7], P30 [SAG1], P35, P41 [GRA4], P54 [ROP2], P66 [ROP1], and P68) in immunoglobulin G (IgG) and IgM recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Following an initial evaluation, six recombinant antigens (P29, P30, P35, P54, P66, and P68) were tested in the IgG and IgM Rec-ELISAs with four groups of samples which span the toxoplasmosis disease spectrum (negative, chronic infection, acute infection, and recent seroconversion). Our results suggest that the combination of P29, P30, and P35 in an IgG Rec-ELISA and the combination of P29, P35, and P66 in an IgM Rec-ELISA can replace the tachyzoite antigen in IgG and IgM serologic tests, respectively. The relative sensitivity, specificity, and agreement for the IgG P29-P30-P35 Rec-ELISA were 98.4, 95.7, and 97.2%, respectively. The resolved sensitivity, specificity, and agreement for the IgM P29-P35-P66 Rec-ELISA were 93.1, 95.0, and 94.5%, respectively. Relative to the tachyzoite-based immunocapture IgM assay, the IgM P29-P35-P66 Rec-ELISA detects fewer samples that contain IgG antibodies with elevated avidity from individuals with an acute toxoplasmosis.
Rapid, Efficient Detection and Drug Susceptibility Testing of
Mycobacterium tuberculosis
in Sputum by Microscopic Observation of Broth Cultures
Authors: Caviedes Luz , Lee Tien-Shun , Gilman Robert H. , Sheen Patricia , Spellman Emily , Lee Ellen H. , Berg Douglas E. , Montenegro-James Sonia ,   The Tuberculosis Working Group in Peru
Published: December 2019

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of
Mycobacterium tuberculosis
(MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases.
Report of the First Human Case of Lobomycosis in the United States
Authors: Burns Robert A. , Roy J. Stephen , Woods Cavan , Padhye Arvind A. , Warnock David W.
Published: December 2019

We describe the first human case of lobomycosis caused by
Lacazia loboi
in a 42-year-old white male resident of Georgia. The patient had traveled to Venezuela 7 years earlier, where he had planned to rappel down Angel Falls in Canaima. Although he never actually rappelled the falls, he did walk under the falls at least three times, exposing himself to the high water pressures of the falls. He noticed a small pustule with surrounding erythema developing on the skin of his right chest wall. The lesion gradually increased in size and had an appearance of a keloid. For cosmetic reasons, the patient sought medical treatment to remove the lesion. After an uncomplicated excision of the lesion, the patient recovered completely. The excised tissue was fixed in formalin for pathologic examination. Tissue sections stained by hematoxylin and eosin, periodic acid-Schiff stain, and Gomori methenamine silver stain procedures showed numerous histiocytes, multinucleated giant cells, and numerous globose or subglobose, lemon-shaped cells producing multiple blastoconidia connected by narrow tube-like connectors and catenate chains of various lengths characteristic of
L. loboi.
Clinical Use of Capillary PCR To Diagnose
Mycoplasma
Pneumonia
Authors: Honda Junichi , Yano Takafumi , Kusaba Mikako , Yonemitsu Junko , Kitajima Hiromoto , Masuoka Megumi , Hamada Kaori , Oizumi Kotaro
Published: December 2019

In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of
Mycoplasma
infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with
Mycoplasma
pneumonia. We performed PCR analysis for
Mycoplasma
DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom
Mycoplasma
pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum
Mycoplasma
antibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing
Mycoplasma
pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results.
Quantitation of Human Immunodeficiency Virus Type 1 RNA in Different Biological Compartments
Authors: Shepard Robin N. , Schock Jody , Robertson Kevin , Shugars Diane C. , Dyer John , Vernazza Pietro , Hall Colin , Cohen Myron S. , Fiscus Susan A.
Published: December 2019

Little information is available describing viral loads in body fluids other than blood. In addition, the suitability of commercially available assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation has not been evaluated in most nonblood fluids. We compared Organon Teknika's nucleic acid sequence-based amplification method (NASBA) and Roche's Amplicor HIV-1 Monitor (reverse transcriptase PCR [RT-PCR]) for quantitating HIV-1 RNA in cerebrospinal fluid (CSF), saliva, breast milk, seminal plasma, and cervical-vaginal lavage fluid (CVL). Saliva and breast milk frequently demonstrated some inhibition in the RT-PCR assay, similar to the inhibition previously described in seminal plasma. Inhibition of the RT-PCR assay was not observed with CSF or CVL, nor in any of the NASBA assays. When fluids from HIV-infected individuals were tested by RT-PCR and NASBA, 73 and 27% of CSF samples and 60 and 40% of breast milk specimens had detectable RNA, respectively. These differences were not statistically significant. In cross-sectional studies using RT-PCR to measure viral RNA in paired blood plasma and CSF samples, 71% of blood plasma samples and 42% of CSF samples were positive. A similar analysis using NASBA with paired blood plasma and CVL, saliva, or seminal plasma samples revealed 91% were blood plasma positive and 55% were CVL positive, 76% were blood plasma positive and 46% were saliva positive, and 83% were blood plasma positive and 63% were seminal plasma positive. NASBA worked fairly well to quantitate HIV-1 RNA from all fluids without apparent inhibition. RT-PCR performed well on CVL and CSF, frequently with greater sensitivity, although its use in other fluids appears limited due to the presence of inhibitors. These studies demonstrate that viral loads in nonblood fluids were generally lower than in blood.
Transmission of
Pneumocystis carinii
DNA from a Patient with
P. carinii
Pneumonia to Immunocompetent Contact Health Care Workers
Authors: Vargas Sergio L. , A. Ponce Carolina , Gigliotti Francis , Ulloa Ana V. , Prieto Susana , Muñoz Maria P. , Hughes Walter T.
Published: December 2019

The transmission of
Pneumocystis carinii
from person to person was studied by detecting
P. carinii
-specific DNA in prospectively obtained noninvasive deep-nasal-swab samples from a child with a documented
P. carinii
pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat
P. carinii
(
P. carinii
f. sp.
carinii
) that amplifies all forms of
P. carinii
and internal primers specific for human
P. carinii
(f. sp.
hominis
).
P. carinii
f. sp.
hominis
DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of
P. carinii
pneumonia, document that person-to-person transmission of
P. carinii
is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk.
Application of a Fluorogenic PCR Assay for Typing and Subtyping of Influenza Viruses in Respiratory Samples
Authors: Schweiger B. , Zadow I. , Heckler R. , Timm H. , Pauli G.
Published: December 2019

A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5′–3′ nuclease activity of the
Taq
DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997–1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998–1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997–1998 and 1998–1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.
A Novel Multiresistant
Streptococcus pneumoniae
Serogroup 19 Clone from Washington State Identified by Pulsed-Field Gel Electrophoresis and Restriction Fragment Length Patterns
Authors: Luna Vicki A. , Jernigan Daniel B. , Tice Alan , Kellner James D. , Roberts Marilyn C.
Published: December 2019

In 1997, a cluster of multiresistant invasive serogroup 19 pneumococcus infections, including two fatalities, was reported in Washington State. Further investigation identified other cases. Fourteen Washington
Streptococcus pneumoniae
isolates, four from Alaska, and eight isolates from eastern Canada with reduced penicillin susceptibility (MIC of ≥1 μg/ml) were included in the study. Pulsed-field gel electrophoresis (PFGE) with
Apa
I,
Sac
II, and
Sma
I restriction enzymes and IS
1167
and
mef
restriction fragment length polymorphism (RFLP) pattern analysis were performed. Twenty of the 26 isolates had identical or related PFGE patterns, with two or all three enzymes, and identical or related IS
1167
RFLP patterns, indicating that they were genetically related. These 20 isolates contained the
mef
gene conferring erythromycin resistance and had identical
mef
RFLP patterns. The PFGE and RFLP patterns were distinct from those of six multiresistant clones previously described and suggest that a new multiresistant clone has appeared in Washington, Alaska, and eastern Canada. This newly characterized clone should be included in the Pneumococcal Molecular Epidemiology Network.
Reverse Cross Blot Hybridization Assay for Rapid Detection of PCR-Amplified DNA from
Candida
Species,
Cryptococcus neoformans
, and
Saccharomyces cerevisiae
in Clinical Samples
Authors: Posteraro Brunella , Sanguinetti Maurizio , Masucci Luca , Romano Lucio , Morace Giulia , Fadda Giovanni
Published: December 2019

A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667–672, 1997), we amplified a fragment of the
ERG11
gene for cytochrome P-450 lanosterol 14α-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the
ERG11
gene of
Candida albicans
(pCal),
C. guilliermondii
(pGui),
C.
(
Torulopsis
)
glabrata
(pGla),
C. kefyr
(pKef),
C. krusei
(pKru),
C. parapsilosis
(pPar),
C. tropicalis
(pTro), the newly described species
C. dubliniensis
(pDub),
Saccharomyces cerevisiae
(pSce), and
Cryptococcus neoformans
(pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples.
Comparison of the Yields of Blood Cultures Using Serum or Plasma from Patients with Early Lyme Disease
Authors: Wormser Gary P. , Bittker Susan , Cooper Denise , Nowakowski John , Nadelman Robert B. , Pavia Charles
Published: December 2019

In an initial experiment, culture-grown
Borrelia burgdorferi
was added to freshly collected uninfected human blood. This in vitro study demonstrated that more spirochetes were distributed into the plasma than into the serum fraction. In a subsequent clinical study,
B. burgdorferi
was recovered from plasma cultures of approximately 50% of 42 patients with early Lyme disease associated with erythema migrans. The rate of recovery from plasma cultures was significantly greater than that from serum cultures (
P
< 0.001).
Evaluation of the Sirscan Automated Zone Reader in a Clinical Microbiology Laboratory
Authors: Medeiros Antone A. , Crellin Joyce
Published: December 2019

We compared readings of Kirby-Bauer plates by the Sirscan, an automated image analyzer that measures zone diameters, to those of experienced clinical microbiologists measuring zones with a hand-held caliper interfaced to a computer and with a ruler. To read plates of
Escherichia coli
,
Morganella morganii
, and
Pseudomonas aeruginosa
containing 12 antibiotic disks the Sirscan took 11 s; technologists took 28 s by caliper and 39 s by ruler. Reading times of four different technologists ranged from 22 to 44 s with the caliper and 10 to 12 s with Sirscan. Upon repeated testing zone size variation rarely exceeded 3 mm by caliper and 1 mm by Sirscan. Over a 4-month period, 368 clinical isolates were tested prospectively by both methods in the Clinical Microbiology Laboratory of the Miriam Hospital. There was good correlation of zone sizes for most antibiotics, but Sirscan zone diameter measurements tended to be 3 to 5 mm larger than caliper readings for ciprofloxacin, norfloxacin, aztreonam, erythromycin, clindamycin, and trimethoprim-sulfamethoxazole. Very major errors (resistant by caliper and susceptible by Sirscan) occurred with 10 of 3,770 readings (0.3%), mainly where breakpoint criteria lacked an intermediate zone. They occurred in testing staphylococci with amoxicillin-clavulanate (5 of 127 isolates, 3.9%), pseudomonas with piperacillin (1 of 28, 3.6%), coagulase-negative staphylococci with oxacillin (2 of 74, 2.7%), gram-negative bacilli with cefuroxime (1 of 209, 0.5%), and mixed species with trimethoprim-sulfamethoxazole (1 of 366, 0.3%). The Sirscan zone reader facilitates accurate, fully quantitative susceptibility testing in clinical microbiology laboratories.
Serologic Diagnosis of Lyme Borreliosis by Using Enzyme-Linked Immunosorbent Assays with Recombinant Antigens
Authors: Magnarelli Louis A. , Ijdo Jacob W. , Padula Steven J. , Flavell Richard A. , Fikrig Erol
Published: December 2019

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of
Borrelia burgdorferi
sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of
B. burgdorferi
except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of
B. burgdorferi
. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.
Detection of Anti-Arboviral Immunoglobulin G by Using a Monoclonal Antibody-Based Capture Enzyme-Linked Immunosorbent Assay
Authors: Johnson Alison J. , Martin Denise A. , Karabatsos Nick , Roehrig John T.
Published: December 2019

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from theAlphavirus,Flavivirus, andBunyavirusgenera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.
Transfer of Erythromycin Resistance from Poultry to Human Clinical Strains ofStaphylococcus aureus
Authors: Khan Saeed A. , Nawaz Mohamed S. , Khan Ashraf A. , Cerniglia Carl E.
Published: December 2019

The transfer ofermAandermCgenes, the two most common resistance determinants of erythromycin resistance, was studied with Luria-Bertani broth in the absence of additional Ca2+or Mg2+ions. Fifteen human and five poultry isolates ofStaphylococcus aureus, which were resistant to erythromycin but carried different genetic markers for erythromycin resistance, were used for conjugation. Since both the donors (Amps-Tetr) and recipients (Ampr-Tets) were resistant to erythromycin, the transconjugants were initially picked up as ampicillin- and tetracycline-resistant colonies. The resistance transfer mechanisms of the chromosomally located erythromycin rRNA methylase geneermAand the plasmid-borneermCgene were monitored by a multiplex PCR and gene-specific internal probing assay. Four groups of transconjugants, based upon the transfer of theermAand/orermCgene, were distinguished from each other by the use of this method. Selective antibiotic screening revealed only one type of transconjugant that was resistant to ampicillin and tetracycline. A high frequency of transfer (4.5 × 10−3) was observed in all of the 23 transconjugants obtained, and the direction of tetracycline and erythromycin resistance marker transfer was determined to be from poultry to clinical isolates. The transfers of theermAandermCgenes were via transposition and transformation, respectively.
Genetic Relatedness of
Salmonella
Isolates from Nondomestic Birds in Southeastern United States
Authors: Hudson Charlene R. , Quist Charlotte , Lee Margie D. , Keyes Kathleen , Dodson Sara V. , Morales Cesar , Sanchez Susan , White David G. , Maurer John J.
Published: December 2019

Salmonella
infections have been implicated in large-scale die-offs of wild birds in the United States. Although we know quite a bit about the epidemiology of
Salmonella
infection among domestic fowl, we know little about the incidence, epidemiology, and genetic relatedness of salmonellae in nondomestic birds. To gain further insight into salmonellae in these hosts, 22
Salmonella
isolates from diseased nondomestic birds were screened for the presence of virulence and antibiotic resistance-associated genes and compared genetically using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA analysis. Of the 22
Salmonella
isolates examined, 15 were positive for the invasion gene
invA
and the virulence plasmid-associated genes
spvC
and
pef
. Most (15 of 22) were generally sensitive to antibiotics. However, two
Salmonella
isolates from pet birds were identified as
Salmonella enterica
serovar Typhimurium DT104. Despite the general susceptibility of these
Salmonella
isolates to most antimicrobial agents, antibiotic resistance-associated genes
intI1
,
merA
, and
aadA1
were identified in a number of these isolates. Five distinct
Xba
I and nine distinct
Bln
I DNA patterns were observed for the 22
Salmonella
isolates typed by PFGE. PFGE analysis determined that
Salmonella
isolates from passerines in Georgia and Wyoming were genetically related.
Bilateral Pulmonary Aspergilloma Caused by an Atypical Isolate of
Aspergillus terreus
Authors: Khan Z. U. , Kortom M. , Marouf R. , Chandy R. , Rinaldi M. G. , Sutton D. A.
Published: December 2019

A case of bilateral pulmonary aspergilloma caused by an atypical isolate of
Aspergillus terreus
is described. The diagnosis was established by the presence of septate, dichotomously branched fungal elements in freshly collected bronchoalveolar lavage and sputum specimens and by repeated isolation of the fungus in culture. Specific precipitating antibodies against the
A. terreus
isolate were demonstrated in the patient's serum. The isolate was atypical as it failed to produce fruiting structures on routine mycological media, but it did so on extended incubation on potato flake agar and produced globose, relatively heavy-walled, hyaline accessory conidia (formerly termed aleurioconidia) on both vegetative and aerial mycelia. Also, it produced an intense yellow diffusing pigment in the medium. The report underscores the increasing importance of
A. terreus
in the etiology of pulmonary aspergillosis. It is suggested that
A. terreus
antigen be included in the battery of serodiagnostic reagents to facilitate the early diagnosis of infections caused by this species.
Isolation of an Unusual
Mycobacterium
Species from an AIDS Patient with Acute Lymphadenitis
Authors: Bajolet Odile , Beguinot Isabelle , Brasme Lucien , Jaussaud Roland , Ingrand Didier , Vincent Véronique
Published: December 2019

A nonchromogenic
Mycobacterium
species was isolated from an AIDS patient with acute lymphadenitis. On the basis of the results of conventional tests, the strain appeared to be an atypical nonphotochromogenic
Mycobacterium kansasii
strain. Sequencing of the 16S rRNA gene revealed a unique nucleic acid sequence, suggesting that the isolate represents an undescribed pathogenic species.
A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle
Authors: Sreevatsan Srinand , Bookout Jack B. , Ringpis Fidel , Perumaalla Veera S. , Ficht Thomas A. , Adams L. Garry , Hagius Sue D. , Elzer Philip H. , Bricker Betsy J. , Kumar Girish K. , Rajasekhar M. , Isloor Srikrishna , Barathur Raj R.
Published: December 2019

A multiplex amplification and detection platform for the diagnosis ofMycobacterium bovisandBrucella abortusinfection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene ofB. abortusand a repeat-sequence region in thehsp65gene ofM. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk forB. abortusand as low as 4 CFU equivalents per ml of milk forM. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected withB. abortus. The assay sensitivity, based on culture status as a “gold standard,” was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding ofB. abortus(samples from India) andM. bovis(samples from Mexico) was identified by BPDA-PCR. In samples from India,B. abortusshedding was identified in 86% of milk ring test-positive animals (n= 15) and 80% of milk ring test-negative cows (n= 5). In samples from Mexico,M. boviswas identified by PCR in 32.6% of pools (n= 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n= 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n= 70) had a modest prevalence ofM. bovisshedding in nasal swabs (2.9%) and milk (1.4%) and ofB. abortusin milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.
No Evidence of Measles Virus in Stapes Samples from Patients with Otosclerosis
Authors: Grayeli Alexis Bozorg , Palmer Pierre , Tran Ba Huy Patrice , Soudant Jacques , Sterkers Olivier , Lebon Pierre , Ferrary Evelyne
Published: December 2019

Otosclerosis is a localized bone dystrophy of unknown etiology mainly involving the stapes. The hypothesis of a persistent infection by the measles virus was based on the inconstant detection of the virus by various methods, including reverse transcription-PCR (RT-PCR) of patients' stapes samples. The aim of this work was to investigate the presence of the measles virus in stapedial otosclerosis foci by different sensitive methods. Pathologic stapes samples were obtained from 35 patients suffering from otosclerosis. Measles virus detection was performed by (i) cocultures of Vero cells and primary cell cultures of bone samples (
n
= 7), (ii) immunofluorescence study of these cocultures (
n
= 3), and (iii) RT-PCR on RNA directly obtained from fresh frozen samples (
n
= 28) and on RNA extracted from the primary cell cultures (
n
= 2). Viral genomic regions coding for N (nucleoprotein) and M (matrix) proteins were separately amplified. PCR sensitivity was optimized on the measles virus Edmonston strain. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as a marker of total RNA recovery. PCR products were tested by Southern blot hybridization technique to improve sensitivity and specificity. PCRs amplifying the M and the N protein genes were able to detect the control measles virus RNA at titers as low as 0.1 and 0.01 50% tissue culture infective dose, respectively. With these highly sensitive methods, we could not evidence the presence of the measles virus in any of our bone samples or primary bone cell cultures. Our results do not confirm the hypothesis of persistent measles virus infection in otosclerosis.
Evaluation of Performance of the Gen-Probe Human Immunodeficiency Virus Type 1 Viral Load Assay Using Primary Subtype A, C, and D Isolates from Kenya
Authors: Emery Sandra , Bodrug Sharon , Richardson Barbra A. , Giachetti Cristina , Bott Martha A. , Panteleeff Dana , Jagodzinski Linda L. , Michael Nelson L. , Nduati Ruth , Bwayo Job , Kreiss Joan K. , Overbaugh Julie
Published: December 2019

Accurate and sensitive quantification of human immunodeficiency virus type 1 (HIV-1) RNA has been invaluable as a marker for disease prognosis and for clinical monitoring of HIV-1 disease. The first generation of commercially available HIV-1 RNA tests were optimized to detect the predominant HIV-1 subtype found in North America and Europe, subtype B. However, these tests are frequently suboptimal in detecting HIV-1 genetic forms or subtypes found in other parts of the world. The goal of the present study was to evaluate the performance of a new viral load assay with non-subtype B viruses. A transcription-mediated amplification method for detection and quantitation of diverse HIV-1 subtypes, called the Gen-Probe HIV-1 viral load assay, is under development. In this study we examined the performance of the Gen-Probe HIV-1 viral load assay relative to that of the commonly used commercial HIV-1 RNA assays using a panel of primary isolates from Kenya. For comparison, we included several subtype B cloned viruses, and we quantified each virus using an in-house quantitative-competitive reverse transcriptase PCR (QC-RT-PCR) method and gag
p24
antigen capture. The Gen-Probe HIV-1 viral load assay and a version of the Roche AMPLICOR HIV-1 MONITOR test (version 1.5) that was designed to detect a broader range of subtypes were both sensitive for the quantification of Kenyan primary isolates, which represented subtype A, C, and D viruses. The Gen-Probe HIV-1 viral load assay was more sensitive for the majority of viruses than the Roche AMPLICOR HIV-1 MONITOR test version 1.0, the Bayer Quantiplex HIV RNA 3.0 assay, or a QC-RT-PCR method in use in our laboratory, suggesting that it provides a useful method for quantifying HIV-1 RNAs from diverse parts of the world, including Africa.
Detection ofLeptospiraspp. in the Aqueous Humor of Horses with Naturally Acquired Recurrent Uveitis
Authors: Faber Nick A. , Crawford Melissa , LeFebvre Rance B. , Buyukmihci Nedim C. , Madigan John E. , Willits Neil H.
Published: December 2019

Leptospiral organisms have long been presumed to be associated with the presence of equine recurrent uveitis. This project was undertaken to determine the presence ofLeptospiraspp. in the aqueous humor of horses with uveitis to determine if there was an association with inflammation. Thirty horses were determined to have recurrent uveitis based on clinical evaluation or history. Sixteen horses were judged clinically and historically to be free of uveitis and were used as controls. Aqueous humor samples were cultured and evaluated by PCR for the presence ofLeptospiraDNA. Serum was collected and evaluated for the presence of antibodies against five serovars in a leptospirosis panel. Twenty-one of 30 horses with recurrent uveitis and one of 16 uveitis-free horses were positive by PCR for the presence ofLeptospiraDNA. Six of these 21 horses with uveitis were culture positive for leptospires from the aqueous humor. Serologic results did not correlate well with the presence ofLeptospiraDNA or organisms in the aqueous humor.Leptospiraspp. are present in a high percentage of horses with naturally occurring recurrent uveitis.
Identification of a Unique
Helicobacter
Species by 16S rRNA Gene Analysis in an Abdominal Abscess from a Patient with X-Linked Hypogammaglobulinemia
Authors: Han S.-R. , Schindel C. , Genitsariotis R. , Märker-Hermann E. , Bhakdi S. , Maeurer M. J.
Published: December 2019

A unique
Helicobacter
species, MZ640285, was isolated from a patient with X-linked hypogammaglobulinemia suffering from recurrent abdominal abscesses and was identified by 16S rRNA gene sequence analysis. In the phylogenetic tree, the isolate fell into a cluster which included
Flexispira rappini
,
Helicobacter bilis
, and
Helicobacter
sp. strain Mainz. Helicobacters are being increasingly recognized as pathogens in immunocompromised hosts. These fastidious bacteria are not easily cultured in the routine diagnostic laboratory, and this is the first report of their identification by 16S rRNA gene sequencing performed directly from a clinical specimen.
Survey for the Presence and Distribution of Human Herpesvirus 8 in Healthy Brain
Authors: Chan Paul K. S. , Ng Ho-Keung , Cheung Jo L. K. , Cheng Augustine F.
Published: December 2019

Brain tissues from both sides of the cerebellum and frontal, temporal, parietal, and occipital lobes were collected postmortem from 30 patients for human herpesvirus 8 (HHV-8) detection by PCR. Overall, 42 of 300 (14.0%) samples were positive, with similar rates for each position. Nineteen patients (63.3%) were positive and showed a significant increase in positivity with age (
P
= 0.007). The results indicate a neuroinvasive and neuropersistent potential of HHV-8.