bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR Assay for Detecting Porcine Cytomegalovirus
Authors: Hamel Andre L. , Lin Lihua , Sachvie Cheryl , Grudeski Elsie , Nayar Gopi P. S.
Published: December 2019

This is the first published report of a PCR assay for detecting porcine cytomegalovirus (PCMV), the causative agent of inclusion body rhinitis in pigs. The DNA to be tested was extracted directly from lungs and nasal scrapings of pigs with various clinical syndromes. Fifty-nine percent (74 of 126) of tested pigs with various clinical syndromes were found to be PCR positive for PCMV. It is hoped that veterinary diagnostic laboratories will benefit by using this PCR assay for routine testing and surveillance of PCMV in pigs.
Development of a Universal Intimin Antiserum and PCR Primers
Authors: Batchelor Miranda , Knutton Stuart , Caprioli Alfredo , Huter Veronika , Zanial Mazlina , Dougan Gordon , Frankel Gad
Published: December 2019

Enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E. coli
(EHEC) constitute a significant risk to human health worldwide. A hallmark of both pathogens is their ability to produce characteristic attaching-and-effacing (A/E) lesions in intestinal epithelial cells. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin, an LEE-encoded bacterial adhesion molecule, mediates the intimate bacterium-host cell interaction characteristic of A/E lesions. On the basis of characterization of the C-terminal 280-amino-acid cell binding domain of intimin (Int280
661–939
), four distinct Int280 types (types α, β, γ, and δ) have been identified. Importantly, Int280α and Int280β antisera specifically recognized their respective intimin types. Using a conserved region of the intimin molecule (Int
388–667
) and primers synthesized to generate the recombinant Int
388–667
, we have now generated universal intimin antiserum and PCR primers that are reactive with the different intimin types expressed by both human and animal A/E lesion-forming strains. Use of immunogold electron microscopy to visualize intimin on the surfaces of EPEC and EHEC strains revealed, in general, a uniform distribution on the bacterial cell surface. However, a filamentous staining pattern was observed with a few strains expressing intimin γ. Cloning of the intimin
eae
gene from one such strain (strain ICC57) into strain CVD206, an EPEC strain which harbors a null deletion in
eae
, produced a uniform intimin staining pattern indicating that, if the filamentous staining pattern defines a filamentous form of intimin γ, it is dependent upon the genetic background of the strain and is not a feature of the intimin molecule.
Species and Genotypic Diversities and Similarities of Pathogenic Yeasts Colonizing Women
Authors: Xu Jianping , Boyd Cynthia M. , Livingston Elizabeth , Meyer Wieland , Madden John F. , Mitchell Thomas G.
Published: December 2019

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 19 women with AIDS, (ii) 11 pregnant women without human immunodeficiency virus (HIV) infection, and (iii) 19 women who were neither pregnant nor infected with HIV. Seven yeast species were recovered, including 59 isolates of
Candida albicans
, 9 isolates of
Candida parapsilosis
, 5 isolates of
Candida krusei
, 3 isolates of
Candida glabrata
, 2 isolates of
Saccharomyces cerevisiae
, and 1 isolate each of
Candida tropicalis
and
Candida lusitaniae
. Seventy unique genotypes were identified by PCR fingerprinting with the M13 core sequence and by random amplified polymorphic DNA analysis. Of the nine shared genotypes, isolates from three different hosts were of one genotype and pairs of strains from different body sites of the same host shared each of the other eight genotypes. Genetic similarities among groups of strains were calculated and compared. We found no significant difference in the patterns of relatedness of strains from the three body sites (oral cavity, vagina, and rectum), regardless of host conditions. The yeast microflora of all three host groups had similar species and genotypic diversities. Furthermore, a single host can be colonized with multiple species or multiple genotypes of the same species at the same or different body sites, indicating dynamic processes of yeast colonization on women.
Bacteriological and Molecular Analysis of Rifampin-Resistant
Mycobacterium tuberculosis
Strains Isolated in Australia
Authors: Yuen Lilly K. W. , Leslie David , Coloe Peter J.
Published: December 2019

To develop a better understanding of the epidemiology and molecular biology of rifampin-resistant
Mycobacterium tuberculosis
strains in Australia, 50 clinical isolates (33 rifampin-resistant and 17 rifampin-sensitive strains) cultured between 1990 and 1997 were analyzed by a number of bacteriological and molecular techniques. Examination of the drug resistance profiles of the 33 rifampin-resistant isolates revealed that 91% were resistant to rifampin in combination with resistance to isoniazid, 88% were resistant to rifampin on first isolation, and 81% showed cross-resistance with rifabutin. On the basis of the demographic data provided for the patients infected with the rifampin-resistant strains, 90% of the patients were born overseas. Of these patients, 64% developed clinical symptoms within 5 years of residence in Australia. On a molecular level, analysis of the
rpoB
gene revealed that 97% of the rifampin-resistant isolates had missense mutations within a conserved region of the gene, and eight types of missense mutations were detected. Of the 31 rifampin-resistant isolates that were typed by restriction fragment length polymorphism (RFLP) analysis, 28 distinct patterns were obtained by RFLP analysis with IS
6110
, and three clusters of genetically related isolates were identified. All isolates within the clusters were from patients who were born overseas and who had the same country of origin. The results from this study provide an overview of the current situation of rifampin resistance in Australia and can serve as a basis for continued monitoring of drug-resistant
M. tuberculosis
strains isolated within the country.
Epidemiology of OropharyngealCandidaColonization and Infection in Patients Receiving Radiation for Head and Neck Cancer
Authors: Redding Spencer W. , Zellars Richard C. , Kirkpatrick William R. , McAtee Robert K. , Caceres Marta A. , Fothergill Annette W. , Lopez-Ribot Jose L. , Bailey Cliff W. , Rinaldi Michael G. , Patterson Thomas F.
Published: December 2019

Oral mucosal colonization and infection withCandidaare common in patients receiving radiation therapy for head and neck cancer. Infection is marked by oral pain and/or burning and can lead to significant patient morbidity. The purpose of this study was to identifyCandidastrain diversity in this population by using a chromogenic medium, subculturing, molecular typing, and antifungal susceptibility testing of clinical isolates. These results were then correlated with clinical outcome in patients treated with fluconazole for infection. Specimens from 30 patients receiving radiation therapy for head and neck cancer were cultured weekly forCandida. Patients exhibiting clinical infection were treated with oral fluconazole. All isolates were plated on CHROMagar Candida and RPMI medium, subcultured, and submitted for antifungal susceptibility testing and molecular typing. Infections occurred in 27% of the patients and were predominantly due toCandida albicans(78%).Candidacarriage occurred in 73% of patients and at 51% of patient visits. Yeasts other thanC. albicanspredominated in carriage, as they were isolated from 59% of patients and at 52% of patient visits. All infections responded clinically, and all isolates were susceptible to fluconazole. Molecular typing showed that most patients had similar strains throughout their radiation treatment. One patient, however, did show the acquisition of a new strain. With this high rate of infection (27%), prophylaxis to prevent infection should be evaluated for these patients.
Molecular Epidemiology and Genetic Diversity of Echovirus Type 30 (E30): Genotypes Correlate with Temporal Dynamics of E30 Isolation
Authors: Oberste M. Steven , Maher Kaija , Kennett Margery L. , Campbell Janice J. , Carpenter Michael S. , Schnurr David , Pallansch Mark A.
Published: December 2019

Echovirus type 30 (E30) (genus,
Enterovirus
; family,
Picornaviridae
) has caused large outbreaks of aseptic meningitis in many regions of the world in the last 40 years. U.S. enterovirus surveillance data for the period 1961 to 1998 indicated that the annual proportion of E30 isolations relative to total enterovirus isolations has fluctuated widely, from a low of 0% in 1966 to a high of 42% in 1998. Peaks of E30 isolations occurred in the years 1968 to 1969, 1981 to 1984, 1990 to 1993, and 1997 to 1998, coincident with large nationwide outbreaks of E30-associated aseptic meningitis. Analysis of the complete VP1 sequence (876 nucleotides) of 136 E30 strains isolated in geographically dispersed regions of the United States and nine other countries between 1956 and 1998 indicated that the currently circulating E30 strains are genetically distinct from those isolated 30 to 40 years ago. Phylogenetic reconstruction demonstrated the existence of at least four distinct genetic groups, three of which have not been isolated in North America since 1981. Two of the three groups disappeared during periods when E30 was isolated infrequently. All North American E30 strains isolated after 1988 were closely related to one another, and all post-1993 isolates were of the same lineage within this group. Surveillance data indicate that E30 causes large national outbreaks of 2- to 4-year durations, separated by periods of relative quiescence. Our results show that shifts in the overall genetic diversity of E30 and the predominant genetic type correlate temporally with the dynamics of E30 isolation. The sequence data also provide a basis for the application of molecular techniques for future epidemiologic investigations of E30 disease.
vanA
and
vanB
Incorporate into an Endemic Ampicillin-Resistant Vancomycin-Sensitive
Enterococcus faecium
Strain: Effect on Interpretation of Clonality
Authors: Suppola Juhana P. , Kolho Elina , Salmenlinna Saara , Tarkka Eveliina , Vuopio-Varkila Jaana , Vaara Martti
Published: December 2019

Clonal spread and horizontal transfer in the spread of vancomycin resistance genes were investigated. Multiplex PCR, pulsed-field gel electrophoresis (PFGE), hybridization of enterococcal plasmids with the
vanA
and
vanB
probes, and sequencing of a fragment of
vanB
were used in the analysis. Before May 1996, 12 vancomycin-resistant
Enterococcus faecium
(VRE) isolates were found in Finland. Between May 1996 and October 1997, 156 VRE isolates were found in the Helsinki area. Between December 1997 and April 1998, fecal samples from 359 patients were cultured for VRE. One new case of colonization with VRE was found. During the outbreak period, 88% (137 of 155) of the VRE isolates belonged to two strains (VRE types I and II), as determined by PFGE. Each VRE type I isolate possessed
vanB
, and five isolates also had
vanA
. Of the 34 VRE type II isolates, 27 possessed
vanA
and 7 possessed
vanB
. Fifteen of 21 (71%) ampicillin-resistant, vancomycin-sensitive
E. faecium
(VSE) isolates found during and after the outbreak period in one ward were also of type II. Two VSE type II isolates were found in the hospital before the outbreak in 1995. By PFGE, the three groups (
vanA
,
vanB
, or no
van
gene) of type II shared the same band differences with the main type of VRE type II with
vanA
. None of the differences was specific to or determinative for any of the groups. Our material suggests that
vanA
and
vanB
incorporate into an endemic ampicillin-resistant VSE strain.
Development of Recombinant Diagnostic Reagents Based on pp85(U14) and p86(U11) Proteins To Detect the Human Immune Response to Human Herpesvirus 7 Infection
Authors: Stefan Alessandra , De Lillo Margherita , Frascaroli Giada , Secchiero Paola , Neipel Frank , Campadelli-Fiume Gabriella
Published: December 2019

Human antibodies raised in response to human herpesvirus 7 (HHV-7) infection are directed predominantly to one or more HHV-7-infected cell proteins with apparent molecular masses of about 85 to 89 kDa. The genes that encode these proteins are unknown. However, several HHH-7 genes that possibly encode proteins in this molecular mass range have been identified. Thus, the proteins encoded by open reading frame U14 (85 kDa) and U11 (86 kDa) were expressed as recombinant proteins in bacteria. Of 13 human serum specimens that recognized the 85- to 89-kDa protein(s) of HHV-7-infected cells by immunoblotting, 12 were also reactive with recombinant pp85(U14) and 8 were reactive with p86(U11). It is concluded that (i) the HHV-7 immunodominant protein is pp85(U14) and (ii) the lack of posttranslational modifications in procaryotically expressed pp85 does not adversely affect the reactivity of human sera. Monoclonal antibody (MAb) 5E1 is an HHV-7-specific MAb directed to pp85(U14). Here, the HHV-7-specific epitope in pp85(U14) was finely mapped to the C′ terminal region between amino acid residues 484 and 502. However, as indicated by the low level of reactivity of human sera with the HHV-7-specific epitope recognized by MAb 5E1, human sera recognize additional epitopes of pp85(U14) that are required for their full reactivity.
rRNA Gene Internal Transcribed Spacer 1 and 2 Sequences of Asexual, Anthropophilic Dermatophytes Related to
Trichophyton rubrum
Authors: Summerbell R. C. , Haugland R. A. , Li A. , Gupta A. K.
Published: December 2019

The ribosomal region spanning the two internal transcribed spacer (ITS) regions and the 5.8S ribosomal DNA region was sequenced for asexual, anthropophilic dermatophyte species with morphological similarity to
Trichophyton rubrum
, as well as for members of the three previously delineated, related major clades in the
T. mentagrophytes
complex. Representative isolates of
T. raubitschekii
,
T. fischeri
, and
T. kanei
were found to have ITS sequences identical to that of
T. rubrum
. The ITS sequences of
T. soudanense
and
T. megninii
differed from that of
T. rubrum
by only a small number of base pairs. Their continued status as species, however, appears to meet criteria outlined in the population genetics-based cohesion species concept of A. R. Templeton. The ITS sequence of
T. tonsurans
differed from that of the biologically distinct
T. equinum
by only 1 bp, while the ITS sequence of the recently described species
T. krajdenii
had a sequence identical to that of
T. mentagrophytes
isolates related to the teleomorph
Arthroderma vanbreuseghemii
.
Bacteremia Due to Extended-Spectrum β-Lactamase-Producing
Escherichia coli
and
Klebsiella pneumoniae
in a Pediatric Oncology Ward: Clinical Features and Identification of Different Plasmids Carrying both SHV-5 and TEM-1 Genes
Authors: Siu L. K. , Lu Po-Liang , Hsueh Po-Ren , Lin F. M. , Chang Shan-Chwen , Luh Kwen-Tay , Ho Monto , Lee Chin-Yun
Published: December 2019

Thirteen patients who had 16 episodes of bacteremia were observed between 1993 and 1997 in a pediatric oncology ward with a high background isolation rate of cefotaxime- or aztreonam-resistant gram-negative bacteria. Four blood isolates were
Escherichia coli
and 12 were
Klebsiella pneumoniae
, and these isolates harbored extended-spectrum β-lactamases (ESBLs). All episodes of bacteremia were nosocomial, all except one of the episodes occurred in neutropenic patients, and all patients were treated with piperacillin or ceftazidime with amikacin and cefazolin prior to the onset of bacteremia. Nine of 13 patients were receiving extended-spectrum β-lactam treatment when the bacteremias caused by ESBL producers occurred. Molecular studies revealed that four
K. pneumoniae
SHV-2-producing isolates from 1994 were of the same clone. Other ESBL producers, including six that carried both TEM-1 and SHV-5, five that carried SHV-5, and one that carried SHV-2 alone, were unrelated. In conclusion, SHV-5 was present in 11 of the 16 isolates and coexisted with TEM-1 in 6 isolates. Acquisition of resistance genes probably occurred under antibiotic selection pressure. This study highlights the importance of routine checks for and detection of ESBL producers. Effective therapy against ESBL producers should be considered early for children who have malignancies and neutropenia and who are septic, despite treatment with a regimen that includes an extended-spectrum β-lactam, in a clinical setting of an increased incidence of ESBL-producing bacteria.
Detection by PCR ofNeospora caninumin Fetal Tissues from Spontaneous Bovine Abortions
Authors: Baszler Timothy V. , Gay Lawrence J. C. , Long Maureen T. , Mathison Bruce A.
Published: December 2019

The routine diagnosis ofNeospora caninumabortion is based upon histopathologic changes in fetal tissues and identification of tissue parasites by immunohistochemistry. Confirmation ofN. caninuminfection by immunohistochemistry has low sensitivity. In the present study, we examined the utility of PCR in detectingN. caninuminfection in fetal tissues from spontaneous bovine abortion. DNA was obtained from fresh and formalin-fixed tissues from 61 bovine fetuses submitted for abortion diagnosis. Histopathology and immunohistochemistry determined the true status ofN. caninuminfection in each fetus. In formalin-fixed paraffin-embedded tissues, PCR detectedN. caninumDNA in 13 of 13 true-positive fetuses (100%) and in 1 of 16 true-negative fetuses (6%). In fresh or frozen tissues, PCR detectedN. caninumDNA in 10 of 13 true-positive fetuses (77%) and 0 of 11 true-negative fetuses (0%). PCR also detectedN. caninumDNA in 6 of 8 fetuses that had typical lesions ofN. caninumbut were immunohistochemistry negative, indicating a higher sensitivity of PCR in comparison to that of immunohistochemistry.N. caninumDNA was amplified most consistently from brain tissue. PCR detection ofN. caninumDNA in formalin-fixed, paraffin-embedded tissues was superior to that in fresh tissues, presumably because of the increased accuracy of sample selection inherent in histologic specimens.
Direct and Rapid Detection by PCR of
Erysipelothrix
sp. DNAs Prepared from Bacterial Strains and Animal Tissues
Authors: Takeshi Kouichi , Makino Souichi , Ikeda Tetsuya , Takada Noriko , Nakashiro Atsushi , Nakanishi Kazunori , Oguma Keiji , Katoh Yoshinobu , Sunagawa Hiroyuki , Ohyama Tohru
Published: December 2019

A PCR method for rapid screening of
Erysipelothrix
spp. in the slaughterhouse was carried out by using four species-specific sets of oligonucleotide primers after initial amplification with the primer set MO101-MO102, which amplifies the 16S rRNA sequences of all four
Erysipelothrix
species. The DNA sequences coding for the rRNA gene cluster, including 16S rRNA, 23S rRNA, and the noncoding region downstream of 5S rRNA, were determined in order to design primers for the species-specific PCR detection system. The homology among the 4.5-kb DNA sequences of the rRNA genes of
Erysipelothrix rhusiopathiae
serovar 2 (DNA Data Bank of Japan accession no.
AB019247
),
E. tonsillarum
serovar 7 (accession no.
AB019248
),
E. rhusiopathiae
serovar 13 (accession no.
AB019249
), and
E. rhusiopathiae
serovar 18 (accession no.
AB019250
) ranged from 96.0 to 98.4%. The PCR amplifications were specific and were able to distinguish the DNAs from each of the four
Erysipelothrix
species. The results of PCR tests performed directly with tissue specimens from diseased animals were compared with the results of cultivation tests, and the PCR tests were completed within 5 h. The test with this species-specific system based on PCR amplification with the DNA sequences coding for the rRNA gene cluster was an accurate, easy-to-read screening method for rapid diagnosis of
Erysipelothrix
sp. infection in the slaughterhouse.
Mutation Patterns of the Reverse Transcriptase and Protease Genes in Human Immunodeficiency Virus Type 1-Infected Patients Undergoing Combination Therapy: Survey of 787 Sequences
Authors: Yahi Nouara , Tamalet Catherine , Tourrès Christian , Tivoli Natacha , Ariasi Franck , Volot Françoise , Gastaut Jean-Albert , Gallais Hervé , Moreau Jacques , Fantini Jacques
Published: December 2019

The aim of the present study was to evaluate the resistance-associated mutations in 302 human immunodeficiency virus type 1 (HIV-1)-infected patients receiving combination therapy and monitored in Marseille, France, hospitals from January 1997 to June 1998. In the reverse transcriptase (RT) gene, the most frequent mutations were found at codons 215 (53%), 41 (34%), and 67, 70, 184, and 210 (>20%). One deletion and two insertions in the β3-β4 hairpin loop of the finger subdomain (codon 69) were detected. Interesting associations and/or exclusions of specific mutations were observed. In 96% of RT genes, a mutation at codon 70 (most frequently, K70R) was associated with a wild-type genotype at position 210 (P< 10−5). Similarly, a mutation at codon 210 (most frequently, L210W) was generally associated with mutations at codons 41 (92%) and 215 (96%) but not at codon 219 (16%) or codon 70 (4%) (P< 10−5). In the protease gene, the most prevalent mutations were at codons 63 (84%), followed by codons 10, 36, 71, 77, and 93 (ca. 20%). As for RT, pairwise associations of mutations were observed. Analysis of the mutation patterns for patients with undetectable HIV-1 loads revealed a high proportion (65%) of wild-type RT genotypes but only 18% wild-type protease genotypes. For patients with high viral loads (>100,000 copies/ml), more than 50% of the RT and protease genes displayed three or more mutations. The significant correlation between the level of viremia in plasma and the number of resistance mutations in the protease (P= 0.007) and RT (P= 0.00078) genes strengthens the importance of defining the genotype of the predominant HIV-1 quasispecies before initiating antiretroviral therapy.
Production of Monoclonal Antibodies Directed against the Microsporidium
Enterocytozoon bieneusi
Authors: Accoceberry Isabelle , Thellier Marc , Desportes-Livage Isabelle , Achbarou Abderrahim , Biligui Sylvestre , Danis Martin , Datry Annick
Published: December 2019

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of
Enterocytozoon bieneusi
and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of
E. bieneusi
, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species
Encephalitozoon intestinalis
or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify
E. bieneusi
spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of
E. bieneusi
. These MAbs have great potential for the demonstration and species determination of
E. bieneusi
, and their application in immunofluorescence identification of
E. bieneusi
in stool samples could offer a new diagnostic tool for clinical laboratories.
Recovery of
Salmonella
by Using Selenite Brilliant Green Sulfa Enrichment Broth
Authors: Chang Chiao-tang , Yuo Chung-Yee , Shen Hui-Ching , Li A-Mai , Chen Chao-yu , Chou Jui-ling , Huang Shiao-ping
Published: December 2019

The efficacy and sensitivity of selenite brilliant green sulfa enrichment (SBG) broth for the isolation of
Salmonella
from fecal specimens were evaluated by using both clinical and artificially infected (artificial) fecal specimens. An examination of 1,588 clinical fecal specimens found
Salmonella
in 296 specimens, including 89 cases detected by the direct-plating xylose-lysine-desoxycholate method and an additional 207 cases detected after enrichment with SBG broth. Therefore, the recovery of
Salmonella
with SBG broth is increased 3.3-fold over that by the direct-plating method alone. Furthermore, the isolation rate of
Salmonella
is higher when using SBG broth than when using gram-negative (GN) broth or GN broth supplemented with sodium selenite. To determine the sensitivity for the recovery of
Salmonella
, artificial specimens containing various amounts of
Salmonella
were prepared and analyzed. The results indicated that the sensitivity is also higher with SBG broth than with GN broth. Moreover, the optimal incubation period for SBG broth can be extended to 24 h. In conclusion, the SBG enrichment method provides a higher recovery rate of
Salmonella
from fecal specimens.
Improved Diagnosis of
Trichomonas vaginalis
Infection by PCR Using Vaginal Swabs and Urine Specimens Compared to Diagnosis by Wet Mount Microscopy, Culture, and Fluorescent Staining
Authors: van der Schee Cindy , van Belkum Alex , Zwijgers Lisette , van der Brugge Esther , O'neill Errol L. , Luijendijk Ad , van Rijsoort-Vos Tineke , van der Meijden Willem I. , Verbrugh Henri , Sluiters Hans J. F.
Published: December 2019

Four vaginal cotton swab specimens were obtained from each of 804 women visiting the outpatient sexually transmitted disease clinic of the Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands, for validation of various forms of
Trichomonas vaginalis
diagnostic procedures. One swab specimen was immediately examined by wet mount microscopy, a second swab was placed in Kupferberg's Trichosel medium for cultivation, and two swabs were placed in phosphate-buffered saline (PBS), pH 7.2. The resulting PBS suspension was used for direct staining with acridine orange and fluorescence microscopy, inoculation of modified Diamond's culture medium, and a PCR specific for
T. vaginalis
. A total of 70 samples positive in one or more of the tests were identified: 31 (3.8%) infections were detected by wet mount microscopy, and 36 (4.4%) were identified by acridine orange staining, as opposed to 40 (4.9%) and 46 (5.7%) positives in modified Diamond's and Trichosel media, respectively. PCR was positive for 61 (7.5%) samples. Secondly, from each of 200 women were obtained a urine sample and a vaginal cotton swab specimen, and 200 urine samples were obtained from men. For the women, 15 (7.4%) of the samples showed a positive result for either the wet mount (
n
= 1), Trichosel culture (
n
= 6), PCR on the vaginal swab sample (
n
= 10), or PCR on the urine specimen (
n
= 11). Four men (2%) were diagnosed with a
T. vaginalis
infection. Thus, PCR appears to be the method of choice for the detection of genital infections with
T. vaginalis.
Mycotic Keratitis Due toCurvularia senegalensisand In Vitro Antifungal Susceptibilities ofCurvulariaspp
Authors: Guarro Josep , Akiti Tiyomi , Horta Roberto Almada- , Morizot Leite-Filho L. A. , Gené Josepa , Ferreira-Gomes Sueli , Aguilar Carme , Ortoneda Montserrat
Published: December 2019

A case of mycotic keratitis due toCurvularia senegalensisis reported. This case represents the third known reported infection caused by this rare species. Fungal hyphae were detected in corneal scrapings, and repeated cultures were positive for this fungi. The patient was presumed cured after a corneal transplant and treatment with itraconazole, but the infection recurred and the patient is waiting for a keratoplasty. The in vitro antifungal susceptibilities of the case strain and another 24 strains belonging to seven species ofCurvulariawere tested for six antifungal agents. With the exception of flucytosine, and occasionally fluconazole, the other drugs assayed (amphotericin B, miconazole, itraconazole, and ketoconazole) were highly effective in vitro.
Lumbar Vertebral Osteomyelitis with Mycotic Abdominal Aortic Aneurysm Caused by Highly Penicillin-Resistant
Streptococcus pneumoniae
Authors: Naktin Jaan , DeSimone Joseph
Published: December 2019

We present a case of vertebral osteomyelitis with an adjacent abdominal aortic mycotic aneurysm caused by a highly penicillin-resistant
Streptococcus pneumoniae
strain. The occurrence of all three phenomena in a single patient has not been previously described. This presentation offers the opportunity to reflect on the increasing incidence of
S. pneumoniae
as a resistant pathogen, the treatment of highly penicillin-resistant
S. pneumoniae
, and the etiologic agents of both vertebral osteomyelitis and mycotic aneurysm.
New PCR Primer Pairs Specific for
Cryptococcus neoformans
Serotype A or B Prepared on the Basis of Random Amplified Polymorphic DNA Fingerprint Pattern Analyses
Authors: Aoki Francisco Hideo , Imai Tamae , Tanaka Reiko , Mikami Yuzuru , Taguchi Hideaki , Nishimura Nancy Fusae , Nishimura Kazuko , Miyaji Makoto , Schreiber Angelica Zaninella , Branchini Maria Luiza Moretti
Published: December 2019

Thirty-three strains of
Cryptococcus neoformans
were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for
C. neoformans
serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respective
C. neoformans
serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from
C. neoformans
serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical
C. neoformans
isolates is discussed.
Flow Cytometric Analysis of Microsporidia Belonging to the Genus
Encephalitozoon
Authors: Moss Delynn M. , Croppo Gian P. , Wallace Sara , Visvesvara Govinda S.
Published: December 2019

Flow cytometry was used in the identification of human microsporidia belonging to the genus
Encephalitozoon
. Microsporidian spores of
Encephalitozoon hellem
,
E. cuniculi
, and
E. intestinalis
were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against
E. hellem
spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-
E. hellem
serum was reactive to
E. hellem
spores but also had cross-reactivity to spores of
E. cuniculi
and
E. intestinalis
. On the other hand, fluorescence histograms showed that rabbit anti-
E. cuniculi
and rabbit anti-
E. intestinalis
sera were reactive with homologous spores only. Monoclonal antibody prepared against
E. hellem
reacted only with spores of
E. hellem
. Neither the polyclonal antibodies nor the monoclonal antibodies reacted with
Cryptosporidium parvum
oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of
Encephalitozoon
were identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of
Encephalitozoon
spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.
Detection of Rubella Virus-Specific Immunoglobulin G in Saliva by an Amplification-Based Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibody to Fluorescein Isothiocyanate
Authors: Vyse A. J. , Brown D. W. G. , Cohen B. J. , Samuel R. , Nokes D. J.
Published: December 2019

An immunoglobulin G (IgG)–capture enzyme-linked immunosorbent assay (ELISA) for rubella virus is described. The assay uses a fluorescein isothiocyanate (FITC)–anti-FITC amplification system. The detection limit of the ELISA was approximately 7 IU of rubella virus-specific IgG per ml of serum sample. For saliva samples the performances of the capture ELISA and previously described radioimmunoassay were assessed, and the results of those two assays were compared to the rubella virus-specific IgG result obtained by a commercial ELISA (Behring Enzygnost) with a panel of paired serum and saliva samples. This comparison showed that the capture ELISA with saliva was more sensitive than the radioimmunoassay and that the results correlated better with the serum IgG result than the results of the radioimmunoassay did, with an overall sensitivity of 82% and a rank correlation of 0.68, whereas the sensitivity and rank correlation for the radioimmunoassay were 74% and 0.45, respectively. For subjects of 10 years of age or younger, the ELISA with saliva had a sensitivity of 94% and a specificity of 100% compared to the results of the ELISA (Behring Enzygnost) for rubella virus-specific IgG with corresponding serum samples. The sensitivity was much lower for subjects ages 17 years or older. The assay may have wider epidemiological use with saliva specimens, particularly those from children.
Quantitation of Human Immunodeficiency Virus Type 2 DNA in Peripheral Blood Mononuclear Cells by Using a Quantitative-Competitive PCR Assay
Authors: Gomes Perpétua , Taveira Nuno Costa , Pereira JoséMoniz , Antunes Francisco , Ferreira M. O. Santos , Lourenço Maria Helena
Published: December 2019

A new quantitative-competitive PCR-based human immunodeficiency virus type 2 (HIV-2) proviral DNA assay (QC-PCR) was developed and used to determine the proviral load in HIV-2-infected individuals. Proviral load varied considerably, with means of 1,831 copies per 10
6
peripheral blood mononuclear cells for asymptomatic subjects (
n
= 19) and 2,587 for AIDS patients (
n
= 2). HIV-2 viral and proviral loads also varied significantly over time in asymptomatic patients. These data suggest that a high level of virus replication occurs throughout the asymptomatic phase of HIV-2 infection.
Gram Type-Specific Broad-Range PCR Amplification for Rapid Detection of 62 Pathogenic Bacteria
Authors: Klausegger Alfred , Hell Markus , Berger Alexandra , Zinober Kerstin , Baier Sabine , Jones Neil , Sperl Wolfgang , Kofler Barbara
Published: December 2019

Broad-range PCR has proven to be useful for the detection of bacteria. A set of broad-range PCR primers directed against conserved regions in the 16S rRNA gene was designed to specifically amplify either gram-positive or gram-negative bacteria. The gram type-specific broad-range PCR correctly classified all 62 pathogenic species tested.
Candida catenulata
Fungemia in a Cancer Patient
Authors: Radosavljevic Mirjana , Koenig Hélène , Letscher-Bru Valérie , Waller Jocelyn , Maloisel Fréderic , Lioure Bruno , Herbrecht Raoul
Published: December 2019

We report the first case of fungemia due to
Candida catenulata
, a contaminant of dairy products.
C. catenulata
was isolated from three blood cultures of a patient with gastric cancer. The patient failed to respond to fluconazole but recovered after treatment was switched to amphotericin B. In vitro,
C. catenulata
was susceptible to amphotericin B and itraconazole and was also susceptible to fluconazole in dose-dependent manner. The likely portal of entry was the digestive tract, as the patient often ate cheese and had multiple gastric ulcerations.
Comparative Analysis and Serovar-Specific Identification of Multiple-Banded Antigen Genes of
Ureaplasma urealyticum
Biovar 1
Authors: Kong Fanrong , Zhu Xuejun , Wang Weizhen , Zhou Xiangzhao , Gordon Susanna , Gilbert Gwendolyn L.
Published: December 2019

Ureaplasma urealyticum
is a causative agent of nongonococcal urethritis and is implicated in the pathogenesis of several other diseases. The species is divided into 14 serovars and two biovars, of which biovar 1 is most commonly isolated from clinical specimens. Reported associations between individual serovars and diseases have been difficult to confirm because of practical difficulties with serotyping. The multiple-banded antigen (MBA) is the predominant
U. urealyticum
antigen recognized during infections in humans and probably has a significant role in virulence. The 5′ end of the MBA gene is relatively conserved but contains biovar, and possibly serovar, specificity. The 5′ ends of the MBA genes of standard strains of
U. urealyticum
biovar 1, consisting of serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and sequenced to identify serovar-specific differences. The 5′ end of the MBA gene sequence of serovar 3 was identical with the previously published sequence and differed by only three bases from that of serovar 14. Significant differences between the MBA gene sequences allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125–UMA269 and UMS-125–UMA269′. Serovars 1 and 6 were distinguished by restriction enzyme analysis of the amplicon and/or by PCR specific for serovar 6. These methods were used to identify and type
U. urealyticum
in 185 (46.3%) of 400 genital specimens from women. Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive specimens. Of 165 specimens containing
U. urealyticum
biovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and 25.5% contained serovars 1, 3/14, and 6, respectively.
U. urealyticum
was found in a significantly higher proportion of pregnant women than in sex workers and other women attending a sexually transmissible diseases clinic (
P
< 0.01). The methods described are relatively rapid, practicable, and specific for serotyping isolates and for direct detection and identification of individual serovars in clinical specimens containing
U. urealyticum
biovar 1.
The
Borrelia burgdorferi
37-Kilodalton Immunoblot Band (P37) Used in Serodiagnosis of Early Lyme Disease Is the
flaA
Gene Product
Authors: Gilmore Robert D. , Murphree Rendi L. , James Angela M. , Sullivan Sarah A. , Johnson Barbara J. B.
Published: December 2019

The 37-kDa protein (P37) of
Borrelia burgdorferi
is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The
P37
gene was cloned from a
B. burgdorferi
genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished in
Escherichia coli
by using a gene construct with the leader peptide deleted and fused to a 38-kDa
E. coli
protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for
B. burgdorferi
anti-P37 reactivity. Lyme disease patient samples serologically negative for the
B. burgdorferi
P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.
Measurement of Urinary Lactoferrin as a Marker of Urinary Tract Infection
Authors: Arao Shinsuke , Matsuura Shiro , Nonomura Mitsuo , Miki Kanji , Kabasawa Keigo , Nakanishi Hisao
Published: December 2019

The usefulness of the measurement of urinary lactoferrin (LF) released from polymorphonuclear leukocytes and of an immunochromatography test strip devised for measuring urinary LF for the simple and rapid diagnosis of urinary tract infections (UTI) was evaluated. Urine specimens were collected from apparently healthy persons and patients diagnosed as suffering from UTI. In the preliminary study, the LF concentrations in 121 normal specimens and 88 specimens from patients (60 with UTI) were quantified by an enzyme-linked immunosorbent assay. The LF concentration was 3,300.0 ± 646.3 ng/ml (average ± standard error of the mean) in the specimens from UTI patients, whereas it was 30.4 ± 2.7 ng/ml and 60.3 ± 14.9 ng/ml in the specimens from healthy persons and the patients without UTI, respectively. Based on these results, a 200-ng/ml LF concentration was chosen as the cutoff value for negativity. Each urine specimen was reexamined with the newly devised immunochromatography (IC) test strip to calculate the indices of efficacy. Based on the cutoff value, it was calculated that the sensitivity, specificity, and positive and negative predictive values of the IC test were 93.3, 89.3, 86.2, and 94.9%, respectively, compared with the results of the microscopic examination of the urine specimens for the presence of leukocytes. The respective indices for UTI were calculated as 95.0, 92.9, 89.7, and 96.6%. The tests were completed within 10 min. These results indicated that urine LF measurement with the IC test strip provides a useful tool for the simple and rapid diagnosis of UTI.
Genetic Diversity and Population Structure of
Vibrio cholerae
Authors: Beltrán Pilar , Delgado Gabriela , Navarro Armando , Trujillo Francisca , Selander Robert K. , Cravioto Alejandro
Published: December 2019

Multilocus enzyme electrophoresis (MLEE) of 397
Vibrio cholerae
isolates, including 143 serogroup reference strains and 244 strains from Mexico and Guatemala, identified 279 electrophoretic types (ETs) distributed in two major divisions (I and II). Linkage disequilibrium was demonstrated in both divisions and in subdivision Ic of division I but not in subdivision Ia, which includes 76% of the ETs. Despite this evidence of relatively frequent recombination, clonal lineages may persist for periods of time measured in at least decades. In addition to the pandemic clones of serogroups O1 and O139, which form a tight cluster of four ETs in subdivision Ia, MLEE analysis identified numerous apparent clonal lineages of non-O1 strains with intercontinental distributions. A clone of serogroup O37 that demonstrated epidemic potential in the 1960s is closely related to the pandemic O1/O139 clones, but the nontoxigenic O1 Inaba El Tor reference strain is not. A strain of serogroup O22, which has been identified as the most likely donor of exogenous
rfb
region DNA to the O1 progenitor of the O139 clone, is distantly related to the O1/O139 clones. The close evolutionary relationships of the O1, O139, and O37 epidemic clones indicates that new cholera clones are likely to arise by the modification of a lineage that is already epidemic or is closely related to such a clone.
Body Lice as Tools for Diagnosis and Surveillance of Reemerging Diseases
Authors: Roux Veronique , Raoult Didier
Published: December 2019

Body lice are vectors of three bacteria which cause human disease:
Rickettsia prowazekii
, the agent of epidemic typhus;
Bartonella quintana
, the agent of trench fever; and
Borrelia recurrentis
, the agent of relapsing fever. A recrudescence of body lice is being observed as the numbers of individuals living under social conditions which predispose individuals to infestation have increased. Because this phenomenon may lead to the reemergence of infections transmitted by body lice, we aimed to assess the occurrence and prevalence of the three agents described above in more than 600 body lice collected from infested individuals in the African countries of Congo, Zimbabwe, and Burundi, in France, in Russia, and in Peru. The presence of the three bacteria in each louse was determined by specific PCR amplification, and the identities of the organisms detected were confirmed by determination of the nucleotide base sequences of the amplification products. Using this approach, we were able to confirm the presence of
R. prowazekii
in lice collected from refugees in Burundi, among whom typhus was epidemic, and the presence of
B. quintana
in lice collected from all locations except the Congo.
B. recurrentis
was never found. Molecular approaches are convenient tools for the detection and identification of bacterial DNA in body lice and for the epidemiological study of louse-borne bacteria from countries where no medical and biological laboratory facilities are available.
Usefulness of PCR and Antigen Latex Agglutination Test with Samples Obtained by Transthoracic Needle Aspiration for Diagnosis of Pneumococcal Pneumonia
Authors: García Amparo , Rosón Beatriz , Pérez José Luis , Verdaguer Ricard , Dorca Jordi , Carratalà Jordi , Casanova Aurora , Manresa Frederic , Gudiol Francesc
Published: December 2019

In a large number of cases, the etiology of community-acquired pneumonia (CAP) is not established. Some cases are probably caused by
Streptococcus pneumoniae
. Transthoracic needle aspiration (TNA) culture has a limited sensitivity which might be improved by antigen detection or gene amplification techniques. We evaluated the capacity of a PCR assay and a latex agglutination test to detect
S. pneumoniae
in samples obtained by TNA from 95 patients with moderate-to-severe CAP. Latex agglutination and PCR had sensitivities of 52.2 and 91.3%, specificities of 88.7 and 83.3%, positive predictive values of 62.3 and 65.6%, and negative predictive values of 83.3 and 96.5%, respectively, when culture techniques were used as the “gold standard.” When we considered expanded criteria for the diagnosis of pneumococcal pneumonia as a standard for our calculations, latex agglutination and PCR had sensitivities of 53.6 and 89.7%, specificities of 93.0 and 90.0%, positive predictive values of 78.9 and 81.3%, and negative predictive values of 80.3 and 94.7%, respectively. The additional diagnosis provided by the PCR assay compared to latex agglutination was 12.2% (95% confidence interval of the difference from 0.4 to 20.1%). PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy (83.3 versus 33.3%). Although PCR is a very sensitive and specific technique, it has not proved to be cost-effective in clinical practice. Conversely, latex agglutination is a fast and simple method whose results might have significant implications for initial antibiotic therapy.