bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients
Authors: Ollert M W , Wende C , Görlich M , McMullan-Vogel C G , Borg-von Zepelin M , Vogel C W , Korting H C
Published: January 2020

The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident. The C. albicans isolates from the HIV-positive group that were characterized by higher levels of proteinase activity were also less susceptible to the widely used azole antifungal ketoconazole and fluconazole. Collectively, the present data are consistent with a concept of early preferential selection of a subpopulation of C. albicans in HIV-infected patients.
Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease
Authors: Quentin R , Huet H , Wang F S , Geslin P , Goudeau A , Selander R K
Published: January 2020

The chromosomal genotypes of 277 isolates of 16 serotypes of Streptococcus agalactiae were characterized by analysis of electrophoretically demonstrable allele profiles at 12 metabolic enzyme loci. The collection comprised the type strain and 276 strains recovered from French symptomatic and asymptomatic subjects. Sixty-one distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Cluster analysis of the ETs revealed two primary phylogenetic divisions separated by a genetic distance of 0.62, Division I contained 67 isolates which could be assigned to 13 ETs. Twenty-seven of these isolates were from samples of cerebrospinal fluid (CSF) from neonatal meningitis patients. Two ETs, separated by a genetic distance of 0.217, contained 26 of these 27 isolates. Division II contained 210 isolates, of which 27 were isolated from CSF. This division was more polymorphic and included 48 ETs. Spanning a genetic distance of 0.3, three clusters and one ET were identified within this group. Twenty-four of 27 strains isolated from CSF belonged to one cluster, and 19 of them belonged to two adjacent ETs with a genetic distance of 0.083. Fifty-five of the 68 serotype Ia strains and 24 of the 26 serotype Ib strains were each confined to one of the evolutionary lineages, and 85 of the 86 strains which carried protein antigen c belonged to phylogenetic division II. Most of the type III organisms were assigned to two clone families. The characteristics of this French population argue for the existence of particular groups of strains responsible for neonatal meningitis and demonstrate that serotyping can supply information about the genetic distribution of strains.
Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by Amplicor PCR
Authors: Moore D F , Curry J I
Published: January 2020

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a PCR-based assay and culture techniques. Results of the PCR assay (Amplicor Mycobacterium tuberculosis Test) were compared with results of standard culture techniques with cultures held for 6 weeks. One thousand nine specimens were included: 301 retrospective specimens (frozen at -70 degrees C and later tested by PCR) and 708 prospective specimens (tested within 1 day of processing). One hundred sixty-two (16%) of the specimens were culture positive for M. tuberculosis; 83 (51%) of these were also fluorochrome stain positive. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 83% (134 of 162 specimens) and 97% (800 of 827 specimens), respectively. The sensitivity for fluorochrome stain-positive specimens was 99%, and that for fluorochrome stain-negative specimens was 66%. The great majority of the 28 PCR-negative, culture-positive specimens were low positives; 27 were smear negative and 19 contained < 100 CFU of M. tuberculosis per ml. The 27 PCR-positive, culture-negative specimens included 24 that were positive by repeat testing by alternate primer PCR and were from patients with tuberculosis on antimicrobial therapy. With these considered culture misses, the final sensitivities of PCR and culture were 85, and 87%, respectively, while the specificities were 99.6 and 100%, respectively. After normal laboratory processing of sputum specimens, the Amplicor PCR assay can be completed in 8 h. Thus, it is possible to have results available within 10 h of specimen submission.
Adenovirus types 2, 8, and 37 associated with genital infections in patients attending a sexually transmitted disease clinic
Authors: Swenson P D , Lowens M S , Celum C L , Hierholzer J C
Published: January 2020

Adenoviruses (Ads) are an important cause of respiratory illness, conjunctivitis, and gastroenteritis, but they are seldom recognized as a potential cause of sexually transmitted disease. We performed virus cultures on approximately 7,000 patients attending a sexually transmitted disease clinic or other health department clinics for the evaluation of genital ulcers, urethritis, or conjunctivitis. Ads were isolated from genital or conjunctival specimens obtained from 23 (0.33%) patients. Among the 20 Ad-positive men, 15 (75%) had urethritis, 12 (60%) had conjunctivitis, and 10 (50%) had both. All three Ad-positive women had vaginal discharge and genital ulcers or fissures. Ad isolates from 17 patients were available for serotyping. Ad type 37 was isolated from 14 patients, Ad type 8 was isolated from 2 patients, and Ad type 2 was isolated from 1 patient. In three of the Ad type 37 cases, Ad was recovered from both urethral and conjunctival specimens. One of the Ad type 8 cases had conjunctivitis, but the Ad type 2 case did not. Ads, particularly type 37, may be a sexually transmissible cause of genital ulcers, urethritis, and conjunctivitis.
Recovery of Mycobacterium avium from cigarettes
Authors: Eaton T , Falkinham J O , von Reyn C F
Published: January 2020

Mycobacterium avium was recovered from tobacco, cigarette paper, and cigarette filters. M. avium could also be recovered from cigarette filters after the cigarettes had been smoked.
Cutaneous phaeohyphomycosis caused by Alternaria longipes in an immunosuppressed patient
Authors: Gené J , Azón-Masoliver A , Guarro J , Ballester F , Pujol I , Llovera M , Ferrer C
Published: January 2020

Alternaria longipes was reported as the agent of a cutaneous infection in a patient with a neoplastic disease. The fungus has not been reported previously as causing disease in humans. It was distinguished by its rather small conidia with smooth or slightly verruculose walls and a pale brown beak which rarely extended into a secondary conidiophore. In vitro inhibitory activities of amphotericin B, ketoconazole, itraconazole, and miconazole were shown.
Molecular subtyping of prevalent M serotypes of Streptococcus pyogenes causing invasive disease
Authors: Stanley J , Linton D , Desai M , Efstratiou A , George R
Published: January 2020

Reproducible methodologies and a scheme for high-resolution genotyping of Streptococcus pyogenes were defined with respect to a study of six predominant M serotypes causing invasive group A streptococcal disease in the United Kingdom. Serotype reference strains were compared with nine clinical isolates of each serotype from patients with diseases such as pneumonia, puerperal sepsis, toxic shock-like-syndrome, cellulitis, or necrotizing fasciitis. Four enzymes were evaluated for their discriminatory power in 16S rRNA gene-specific ribotyping. Discriminatory power was greatest with EcoRI, which generated serotype-specific ribotypes, and with SacI, which could subdivide strains of the same M serotype. Twenty-five combined ribotypes were found among the 60 strains, and the indices of discriminatory power (D values) of this method varied from 0.51 within serotype M1 to 0.98 within strains of serotype M5. Macrorestriction with the rarely cutting endonuclease SmaI and pulsed-field gel electrophoresis gave D values varying from 0.37 within serotype M1 to the maximal 1.0 within serotype M5. Comparison of macrorestriction profiles revealed various degrees of genetic heterogeneity within M serotypes. Strains of M1, M3, M6, and M11 exhibited clonally related macrorestriction profiles, while those of R28 and M5 strains were consistent with polyphyletic origin.
Detection of newly recognized rodent parvoviruses by PCR
Authors: Besselsen D G , Besch-Williford C L , Pintel D J , Franklin C L , Hook R R , Riley L K
Published: January 2020

Several autonomous parvovirus isolates distinct from the prototypic rodent parvoviruses have recently been identified. These include variants of a mouse orphan parvovirus (MOPV) and a hamster isolate designated hamster orphan parvovirus (HOPV). In this study, a PCR primer set specific for these newly identified rodent parvoviruses was designed on the basis of DNA sequence comparisons of these isolates with other autonomous parvoviruses. The specificity of the primer set was determined by testing viral preparations of seven different parvoviruses and eight other viruses known to infect rodents. The PCR assay amplified the expected 260-bp product only in the presence of DNA from MOPV, HOPV, or LuIII a parvovirus of unknown species origin. The assay was able to detect as little as 10 pg of MOPV viral DNA or 1 pg of HOPV viral DNA, and it was able to detect MOPV in tissues from naturally infected mice and HOPV in tissues from experimentally infected hamsters. In contrast, the 260-bp product was not amplified from tissues of MOPV-negative mice or mock-infected hamsters. Our findings indicate that this PCR assay provides a rapid, specific, and sensitive method for the detection of MOPV in mice, HOPV in hamsters, and MOPV and HOPV in cell culture systems and that it may also be useful for the detection of LuIII contamination of cell culture systems.
Escherichia coli O157:H7 restriction pattern recognition by artificial neural network
Authors: Carson C A , Keller J M , McAdoo K K , Wang D , Higgins B , Bailey C W , Thorne J G , Payne B J , Skala M , Hahn A W
Published: January 2020

An artificial neural network model for the recognition of Escherichia coli O157:H7 restriction patterns was designed. In the training phase, images of two classes of E. coli isolates (O157:H7 and non-O157:H7) were digitized and transmitted to the neural network. The system was then tested for recognition of images not included in the training set. Promising results were achieved with the designed network configuration, providing a basis for further study. This application of a new generation of computation technology serves as an example of its usefulness in microbiology.
Geographical clustering of human T-cell lymphotropic virus type 1 infection in Honduras
Authors: de Rivera I L , Amador L , Mourra S , Li Z , Rasheed S
Published: January 2020

Geographical clustering of human T-cell lymphotropic virus type 1 (HTLV-1) infection has been identified in the nonmestizo communities in several cities along the Atlantic coast of Honduras. Of the 2,651 serum samples tested, 122 samples were repeatedly reactive for HTLV-1 antibodies in two different enzyme immunoassays and 3 were indeterminate. These sera did not react in the HTLV-2-specific antibody tests. The presence of HTLV-1 antibodies was confirmed by HTLV-1 immunoblots or Western blots (immunoblots), and the infection was verified by the detection of HTLV-1-specific genetic sequences in the cellular DNA by PCR. Genomic DNA from the peripheral blood mononuclear cells was first tested with generic primers and probes that identified both HTLV-1 and HTLV-2. Next, all DNA samples that showed HTLV reactivity were tested by PCR with specific primers and probes that distinguished HTLV-1 sequences from those of HTLV-2. Our results indicate that only HTLV-1 infection was present in the blood of both mestizo and nonmestizo residents of 15 cities in the Republic of Honduras. The overall prevalence of HTLV-1 infection in the nonmestizo population was 8.1% (95% confidence limit, 6.6 to 9.7%). The mestizo population residing in the same geographical vicinities showed a HTLV-1 antibodies in 0.5% of serum samples tested (95% confidence limit, 0.6 to 1.7%), indicating a significantly greater prevalence of HTLV-1 infection in the nonmestizo population than in the mestizo ethnic groups living in Honduras (P = 0.0001). Since no HTLV-2 antibody reactivity or HTLV-2-specific genetic sequences were detected by PCR with different primers and probes, it was concluded that HTLV-2 infection was not present in the Honduran population groups we tested. Our study also suggested an endemic nature for this virus because there was no difference in the prevalence rate of HTLV-1 antibodies in the nonmestizo community living in the coastal towns of Honduras between 1989 and 1993. This is the first report of HTLV-1 cluster identification in Honduras, Central America.
Genomic study of Rickettsia akari by pulsed-field gel electrophoresis
Authors: Eremeeva M , Balayeva N , Ignatovich V , Raoult D
Published: January 2020

Pulsed-field gel electrophoresis of SmaI-, EagI-, and BssHII-digested DNA was used to perform restriction fragment length polymorphism analysis of Rickettsia akari strains isolated from humans, rodents, and mites in the United States and Ukraine. Although some differences in biological and serological characteristics were present between strains, the genomic studies demonstrated a high degree of intraspecies homogeneity of R. akari isolates. Our results confirm the value of pulsed-field gel electrophoresis-restriction fragment length polymorphism analysis for the identification of species of rickettsiae.
Comparison of SHARP signal system and Southern blot hybridization analysis for detection of cytomegalovirus in clinical specimens by PCR
Authors: Espy M J , Smith T F
Published: January 2020

Thirty cytomegalovirus cell culture-positive samples were tested by the SHARP Signal System. Twenty-seven specimens (100% agreement) were identified by both methods. The SHARP Signal System is rapid (4 h), easy to perform, and potentially adaptable to automation.
Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens
Authors: Mahony J B , Luinstra K E , Tyndall M , Sellors J W , Krepel J , Chernesky M
Published: January 2020

We developed a multiplex PCR (M-PCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. M-PCR employed C. trachomatis-specific primers KL1-KL2 and N. gonorrhoeae-specific primers HO1-HO3 and produced products of 241 and 390 bp, respectively. PCR products were easily detected by agarose gel electrophoresis and confirmed by Southern hybridization using labelled oligonucleotide probes. M-PCR had a sensitivity of 10 fg of C. trachomatis and N. gonorrhoeae DNA (equivalent to 1 to 2 genome copies). M-PCR detected the presence of C. trachomatis and N. gonorrhoeae DNA in 15 male urethral and 12 female endocervical specimens, 3 of which were positive for C. trachomatis, 18 of which were positive for N. gonorrhoeae and 6 of which were positive for both organisms. M-PCR was evaluated further by testing 200 male first void urine (FVU) specimens, of which 18 were positive by C. trachomatis PCR and Chlamydiazyme and 4 were positive by C. trachomatis PCR but negative by Chlamydiazyme. All 22 FVU specimens were positive by a confirmatory PCR using a second plasmid target and were positive by M-PCR. Ten of 11 men with cultures that were positive for N. gonorrhoeae had FVU specimens that were positive by both N. gonorrhoeae PCR and M-PCR. Two other men with negative N. gonorrhoeae urethral cultures had FVU specimens that were positive by N. gonorrhoeae PCR, by two confirmatory N. gonorrhoeae PCR assays using 165 rRNA and cytosine methyltransferase primers, and by M-PCR. The sensitivity of M-PCR for detecting C. trachomatis was 100% (22 of 22 specimens), compared with 81.8% (18 of 22 specimens) for enzyme immunoassay. Sensitivity of M-PCR for N. gonorrhoeae was 92.3% (12 of 13 specimens) compared with 84.6% (11 of 13 specimens) for urethral culture. The specificity of M-PCR was 100% for both C. trachomatis (178 of 13 specimens) and N. gonorrhoeae (187 of 187 specimens). M-PCR testing of FVU specimens provided a sensitive and noninvasive method for detecting C. trachomatis and N. gonorrhoeae infection in men.
Evaluation of sampling sites for detection of upper respiratory tract carriage of Streptococcus pneumoniae and Haemophilus influenzae among healthy Filipino infants
Authors: Capeding M R , Nohynek H , Sombrero L T , Pascual L G , Sunico E S , Esparar G A , Esko E , Leinonen M , Ruutu P
Published: January 2020

Two sampling techniques, nasal swabbing and oropharyngeal swabbing, for detection of the upper respiratory tract carriage of Streptococcus pneumoniae and Haemophilus influenzae were studied prospectively with 296 healthy Filipino infants at various ages: 6 to 8, 10 to 12, 14 to 17, 18 to 22, 32 to 39, and 46 to 65 weeks. In all age groups S. pneumoniae was isolated significantly more often (P < 0.0001) from the nasal site than from the oropharyngeal site. H. influenzae was found equally often at both sites.
Ligase chain reaction for detection of Neisseria gonorrhoeae in urogenital swabs
Authors: Ching S , Lee H , Hook E W , Jacobs M R , Zenilman J
Published: January 2020

The ligase chain reaction (LCR) is an in vitro nucleic acid amplification technique that exponentially amplifies targeted DNA sequences. In a multicenter study, we evaluated the use of a 4-h LCR-based assay for the diagnosis of Neisseria gonorrhoeae infection of the cervix and male urethra. The LCR results were compared with those of culture for N. gonorrhoeae by using selective media. This assay amplifies target sequences within the N. gonorrhoeae opacity gene. Discordant LCR-positive and culture-negative specimens were further evaluated by testing by another LCR assay which used N. gonorrhoeae-specific pilin probe sets. A total of 1,539 female endocervical specimens and 808 male urethral swab specimens were evaluated in the study. An expanded "gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive specimens. After resolution of discrepant samples, the sensitivities of the N. gonorrhoeae LCR assays for the female and male specimens were 97.3 and 98.5%, respectively, with specificities of 99.6 and 99.8%, respectively. Resolved culture sensitivities were 83.9 and 96.5% for the female and male specimens, respectively. The LCR assay for gonorrhea is a rapid, highly sensitive nonculture method for detecting gonococcal infection of the cervix and male urethra.
Characterization of Aeromonas trota strains that cross-react with Vibrio cholerae O139 Bengal
Authors: Albert M J , Ansaruzzaman M , Shimada T , Rahman A , Bhuiyan N A , Nahar S , Qadri F , Islam M S
Published: January 2020

It has previously been shown that Vibrio cholerae O139 Bengal shares antigens with V. cholerae serogroups O22 and O155. We detected six surface water isolates of Aeromonas trota that agglutinated in polyclonal antisera to V. cholerae O139 and V. cholerae O22 but not in antiserum to V. cholerae O155. On the basis of agglutinin-absorption studies, the antigenic relationship between the cross-reacting bacteria were found to be in an a,b-a,c fashion, where a is the common antigenic epitope and b and c are unique epitopes. The antigen sharing between A. trota strains and V. cholerae O139 was confirmed in immunoblot studies. However, A. trota strains did not react with two monoclonal antibodies specific for V. cholerae O139 and, consequently, tested negative in the Bengal SMART rapid diagnostic test for V. cholerae O139 which uses one of the monoclonal antibodies. A polyclonal antiserum to a cross-reacting A. trota strain cross-protected infant mice against cholera on challenge with virulent V. cholerae O139. All A. trota strains were cytotoxic for HeLa cells, positive for adherence to HEp-2 cells, and weakly invasive for HEp-2 cells; one strain was heat-stable toxin positive in the suckling mouse assay; however, all strains were negative for cholera toxin-like enterotoxin. Studies on bacteria that share somatic antigen with V. cholerae O139 may shed further light on the genesis of V. cholerae O139.
PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples
Authors: Kox L F , van Leeuwen J , Knijper S , Jansen H M , Kolk A H
Published: January 2020

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.
Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia
Authors: van Soolingen D , Qian L , de Haas P E , Douglas J T , Traore H , Portaels F , Qing H Z , Enkhsaikan D , Nymadawa P , van Embden J D
Published: January 2020

Analysis of the population structure of Mycobacterium tuberculosis strains from the People's Republic of China showed that the vast majority belong to a genetically closely related group. These strains shared the majority of their IS6110 DNA-containing restriction fragments, and also, the DNA polymorphism associated with other repetitive DNA elements, like the polymorphic GC-rich sequence and the direct repeat, was very limited. Because the majority of these strains originated from the province of Beijing, we designated this grouping the "Beijing family" of M. tuberculosis strains. Strains of this family were also found to dominate in neighboring countries such as Mongolia, South Korea, and Thailand, whereas a low prevalence of such strains was observed in countries on other continents. These data indicate that strains of the Beijing family recently expanded from a single ancestor which had a selective advantage. It is speculated that long-term Mycobacterium bovis BCG vaccination may be one of the selective forces implicated in the successful spread of the Beijing genotype.
Transhemispheric exchange of Lyme disease spirochetes by seabirds
Authors: Olsen B , Duffy D C , Jaenson T G , Gylfe A , Bonnedahl J , Bergström S
Published: January 2020

Lyme disease is a zoonosis transmitted by ticks and caused by the spirochete Borrelia burgdorferi sensu lato. Epidemiological and ecological investigations to date have focused on the terrestrial forms of Lyme disease. Here we show a significant role for seabirds in a global transmission cycle by demonstrating the presence of Lyme disease Borrelia spirochetes in Ixodes uriae ticks from several seabird colonies in both the Southern and Northern Hemispheres. Borrelia DNA was isolated from I. uriae ticks and from cultured spirochetes. Sequence analysis of a conserved region of the flagellin (fla) gene revealed that the DNA obtained was from B. garinii regardless of the geographical origin of the sample. Identical fla gene fragments in ticks obtained from different hemispheres indicate a transhemispheric exchange of Lyme disease spirochetes. A marine ecological niche and a marine epidemiological route for Lyme disease borreliae are proposed.
Random amplified polymorphic DNA analysis of clinically and environmentally isolated Cryptococcus neoformans in Nagasaki
Authors: Yamamoto Y , Kohno S , Koga H , Kakeya H , Tomono K , Kaku M , Yamazaki T , Arisawa M , Hara K
Published: January 2020

We examined clinical (pulmonary cryptococcosis and cryptococcal meningitis) and environmental (pigeon excreta) isolates of Cryptococcus neoformans var. neoformans (serotype A) in the southern Japanese prefecture of Nagasaki. The random amplified polymorphic DNA profiles obtained by using three primers revealed six patterns among 21 clinical isolates and three patterns among 8 environmental isolates. Pattern I was the most common (18 of 29 isolates) and was found among isolates obtained throughout the entire Nagasaki Prefecture. Patterns I, III, and IV were found among both clinical and environmental isolates. Patterns I and IV had a characteristic distribution, and in particular, pattern IV was isolated exclusively (five of six isolates) from isolates from Nagasaki City. Two environmental isolates from two locations associated strongly with two patients revealed identical random amplified polymorphic DNA patterns (patterns I and IV) for isolates from each patient. Our results suggest that clinical and environmental isolates belong to the same pool of C. neoformans isolates and that these isolates have certain geographic locations, although the number of isolated strains was limited.
Solid-phase C1q-directed bacterial capture followed by PCR for detection of Chlamydia trachomatis in clinical specimens
Authors: Herbrink P , van den Munckhof H A , Niesters H G , Goessens W H , Stolz E , Quint W G
Published: January 2020

An antigen capture system based on the binding of bacteria to solid-phase immobilized complement C1q followed by PCR for detection of Chlamydia trachomatis in clinical samples was developed and clinically evaluated. Comparison of C1q-directed antigen capture PCR with cell culture and direct PCR on 71 consecutive clinical specimens revealed an identical sensitivity. In this group, all 11 cell culture-positive samples were positive by direct PCR and C1q-directed antigen capture PCR. In addition, two samples found negative by cell culture were found positive by both direct PCR and C1q-directed antigen capture PCR. To further assess the sensitivity of C1q-directed antigen capture PCR, 20 clinical samples with one to five inclusions in cell culture and 20 clinical samples with 6 to 20 inclusions in cell culture were tested. Results obtained showed sensitivities of 95 and 90% for clinical samples with 6 to 20 and 1 to 5 inclusions in cell culture, respectively. Using C1q-coated solid phases, C1q-binding Chlamydia particles can be concentrated from large volumes with concomitant removal of inhibitors of PCR, allowing the use of large volumes of clinical samples for clinical testing. Since C1q has been shown to bind to a range of gram-negative bacteria, the newly developed technique has utility for a broad range of bacteria.
Mycobacterial testing in clinical laboratories that participate in the College of American Pathologists' Mycobacteriology E survey: results of a 1993 questionnaire
Authors: Woods G L , Witebsky F G
Published: January 2020

Participants in the College of American Pathologists' Mycobacteriology E proficiency testing survey in 1993 were asked to complete a questionnaire addressing mycobacterial test methods, test volume, and frequency of detection of drug-resistant Mycobacterium tuberculosis (MTB). A similar questionnaire had been distributed in 1992. The population responding to the 1993 questionnaire changed, because of a shift of small hospitals to the limited Mycobacteriology E1 survey, and the format of some questions was altered, so a direct comparison of 1992 and 1993 responses was not always possible. Among participants who answered the questions in both years, there was a significant increase in the use of the fluorochrome stain (57% in 1992, 61% in 1993), BACTEC TB for culture (34% in 1992, 38% in 1993) and susceptibility testing (51% in 1992, 61% in 1993), and DNA probes for identification (30% in 1992, 51% in 1993). The percentage of participants who processed respiratory specimens at least seven times per week increased from 9% in 1992 to 13% in 1993, and the percentage processing five times per week increased from 68 to 72%. The percentage of respondents who reported an identification of MTB within 21 days of specimen receipts and susceptibility test results within 28 days in 1992 and 1993 increased from 30 to 41% and from 12 to 19%, respectively. In regard to resistant MTB, 177 institutions in 1991 and 291 in 1992 reported resistance to isoniazid, and 114 in 1991 and 187 in 1992 reported resistance to both isoniazid and rifampin. Laboratorians are to be applauded for using the more rapid mycobacterial testing methods; however, given that tuberculosis remains a problem, this trend must continue.
In vitro growth of the microsporidian Septata intestinalis from an AIDS patient with disseminated illness
Authors: Doultree J C , Maerz A L , Ryan N J , Baird R W , Wright E , Crowe S M , Marshall J A
Published: January 2020

A new species of microsporidian, Septata intestinalis, was recently recognized as an opportunistic pathogen of AIDS patients. In this study, it was cultured from the nasopharyngeal aspirate of a human immunodeficiency virus type 1-infected patient with disseminated microsporidiosis. In human embryonic lung cells exposed to S. intestinalis, a cytopathic effect appeared within 28 days as foci of rounded up cells. Thin-section electron microscopy showed a variety of developmental stages of the microsporidium within parasitophorous vacuoles. In monocyte-derived macrophages, evidence of infection and development of the parasite was demonstrated by light and electron microscopy. In both infected human embryonic lung cells and monocyte-derived macrophages, a network of septa separated individual spores. Partial sequencing of the RNA small-subunit gene (16S rDNA gene) confirmed the identity of this parasite as S. intestinalis. This is the first report of the isolation of S. intestinalis in vitro and provides evidence that the parasite can be disseminated by macrophages.
Phenotypic characteristics, fatty acid composition, and isoprenoid quinone content of CDC group IIg bacteria
Authors: Hollis D G , Daneshvar M I , Moss C W , Baker C N
Published: January 2020

Eleven strains of eugonic, nonoxidative, gram-negative rods isolated from clinical specimens formed a distinct group that was designated CDC group IIg. Five of the 11 isolates were from wounds. The phenotypic characteristics of CDC group IIg were most similar to those of Weeksella species, with the major difference being that CDC group IIg strains grew on MacConkey agar in 1 to 2 days, did not hydrolyze gelatin, and did not produce urease. All 11 strains of CDC group IIg possessed a distinct fatty acid profile that was characterized by large amounts (19 to 29%) of 18:1 omega 7c, 16:0, and 16:1 omega 7c, moderate amounts (6 to 10%) of 3-OH-14:0 and 14:0, and smaller amounts (1 to 2%) of 18:2, 18:0, and 3-OH-16:0. This fatty acid profile differs from those of Weeksella species by the absence of branched-chain fatty acids. CDC group IIg contains ubiquinone-8, as opposed to menaquinone-6 in Weeksella species. The isolates were susceptible to a variety of antimicrobial agents, including the aminoglycosides, tetracyclines, quinolones, sulfonamides, and polymyxin B.
Difficulties encountered in identification of a nutritionally deficient streptococcus on the basis of its failure to revert to streptococcal morphology
Authors: Bottone E J , Thomas C A , Lindquist D , Janda J M
Published: January 2020

Difficulties were encountered in the recognition of a nutritionally deficient streptococcus which continued to display aberrant morphologic forms (especially bulbous swellings and filament formation) despite provision of growth factors. With isolates displaying diverse morphologic entities not characteristic of a given species, e.g., Streptobacillus moniliformis or Erysipelothrix rhusiopathiae, nutritionally deficient streptococci should be considered.
Evaluation of CPS ID2 medium for detection of urinary tract bacterial isolates in specimens from a rehabilitation center
Authors: Mazoyer M A , Orenga S , Doleans F , Freney J
Published: January 2020

CPS ID2 medium (bioMérieux) enables the presumptive identification of Escherichia coli and enterococci as well as the detection of indologenous or nonindologenous Proteeae and bacteria belonging to the Klebsiella, Enterobacter, and Serratia group with a specificity ranging from 98 to 100%. When the cultures were polymicrobial, the sensitivity varied from 70 to 97%, and the sensitivity varied from 97 to 100% when they were monomicrobial.
Case report of and description of parasite in Mammomonogamus laryngeus (human syngamosis) infection
Authors: Nosanchuk J S , Wade S E , Landolf M
Published: January 2020

About 100 cases of human infections with Mammomonogamus laryngeus (Syngamidae, Syngaminae) have been reported, with virtually all cases originating in the Caribbean Islands and Brazil. This report describes the fifth patient in North America infected with M. laryngeus and the first case documented to originate in Jamaica. The patient complained of a characteristic persistent nonproductive cough and a lump in her throat. M. laryngeus is a nematode in which the male and female are permanently joined in copula, producing a distinctive Y shape. Since there is scant clinical information about this parasite, this report includes a description of the adult worms and eggs, a summary of the epidemiology, and the clinical manifestations in humans. Illustrations are presented to facilitate future identification of this parasite and to expedite treatment.
Multiplex PCR assay and simple preparation method for stool specimens detect enterotoxigenic Escherichia coli DNA during course of infection
Authors: Stacy-Phipps S , Mecca J J , Weiss J B
Published: January 2020

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among travelers and residents of developing countries. ETEC produces either a heat-stable toxin or a heat-labile toxin, or both, encoded by plasmid-borne ST and LT genes, respectively. Diagnosis of infection with this subclass of E. coli can be performed with oligonucleotide hybridization probes; however, the sensitivity and specificity of this method are insufficient. A nonradioactive multiplex PCR assay that provides a sensitive and specific method for detecting the presence of either or both toxin genes has been developed. A simple procedure that removed inhibitors of the PCR while efficiently releasing ETEC DNA from stool specimens for subsequent amplification was used. The results for samples from a human volunteer study of ETEC infection indicated that this method of sample preparation results in greater clinical sensitivity than conventional total nucleic acid extraction and ethanol precipitation. Detection of ETEC by a multiplex PCR assay in stool specimens directly processed with a glass matrix and chaotropic solution had greater sensitivity than culture.
Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences
Authors: Dolzani L , Tonin E , Lagatolla C , Prandin L , Monti-Bragadin C
Published: January 2020

Members of the genus Acinetobacter are reported to be involved in hospital-acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested. This study thus proposes the detection of restriction length polymorphism in the spacer sequences between the 16S and 23S rRNA genes as a method for the identification of isolates in the A. calcoaceticus-A. baumannii complex.
Impact of free verotoxin testing on epidemiology of diarrhea caused by verotoxin-producing Escherichia coli
Authors: Ramotar K , Henderson E , Szumski R , Louie T J
Published: January 2020

During a 10-week period in the summer of 1990, an epidemiologic investigation of the prevalence of verotoxin (VT)-producing Escherichia coli infection was conducted in Calgary, Alberta, Canada. Consecutive stool specimens (n = 3,577) were cultured for E. coli O157:H7, and fecal filtrates were tested for free VTs (FVTs). E. coli O157:H7 was recovered from 22 specimens (0.6%), but VT was detected in 74 specimens (2.1%). Sixty-nine stool specimens positive for FVTs or E. coli O157:H7 were probed for VT genes by colony blot hybridization; 22 of 38 VT gene probe-positive isolates were non-O157:H7 E. coli organisms. Fourteen of 22 strains could not be induced to produce VT in vitro, despite the presence of FVTs in the stool sample, positivity on colony blot hybridization, positive PCR probes with the primers described by Pollard et al. (D. R. Pollard, W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee, J. Clin. Microbiol. 28:540-545, 1990) or Gannon et al. (V. P. Gannon, R. K. King, J. Y. Kim, and E. J. Golsteyn-Thomas, Appl. Environ. Microbiol. 58:3809-3815, 1992) (but not those described by Karch and Meyer [H. Karch and T. Meyer, J. Clin. Microbiol. 27:2751-2757, 1989]), and positive Southern blot analysis of isolates in 10 of 14 strains. The patient survey questionnaire showed that E. coli O157:H7 infection was associated with bloody diarrhea of short duration, whereas infection with other serotypes or persistence of FVT only was associated with longer-duration nonbloody diarrheal illness. We conclude that (i) detection of FVT in stools enhances the diagnosis of VT infection threefold over cultures for E. coli O157:H7, (ii) cultures for E.coli O157:H7 detect the majority of organisms of that serotype, (iii) the spectrum of disease produced by organisms of non-O157:H7 serotypes may include less severe but more protracted illness, and (iv) differences in the in vivo and in vitro expression of toxin and results of genetic probe studies highlight the need to examine control mechanisms of toxin production.
Cost-effective, clinically relevant method for rapid identification of beta-hemolytic streptococci and enterococci
Authors: Kirby R , Ruoff K L
Published: January 2020

Currently popular agglutination and coagglutination methods for the identification of beta-hemolytic streptococci, although rapid and simple to perform, are costly. Furthermore, they fail to distinguish between clinically relevant species and normal flora of the same serogroup. We investigated the use of a series of four physiologic tests to differentiate beta-hemolytic streptococci and enterococci into five clinically relevant groups. We also investigated the use of a new product, Visi-Spot, and evaluated an alternate method for the detection of beta-D-glucuronidase production. Our results suggest that for most routine processing of beta-hemolytic streptococci, physiologic tests are sufficiently rapid, more accurate, and far less costly to perform than serologic methods. The facility of our scheme is enhanced by the use of the Visi-Spot test and the substitution of a commercially available product for more traditional methods of detecting beta-D-glucuronidase.
Typing and subtyping of influenza viruses in clinical samples by PCR
Authors: Wright K E , Wilson G A , Novosad D , Dimock C , Tan D , Weber J M
Published: January 2020

Type A and B influenza viruses can cause a wide spectrum of illness, and these viruses are responsible for considerable mortality and morbidity. Rapid typing of isolates is desirable when amantadine treatment or prophylaxis of contacts of type A influenza virus carriers is considered, but the available rapid techniques lack sensitivity and standard diagnostic methods require expansion of virus in tissue culture or embryonated hens' eggs. We developed a series of oligonucleotide primers able to detect, type, and subtype type A influenza viruses in a single reverse transcription-PCR. RNA was isolated from clinical specimens, and cDNA was generated with random primers. PCR was carried out with a mixture of primers specific for influenza viruses of types B, A/H1 and A/H3, and subtyping of the neuraminidase was carried out on the same cDNA template under identical conditions. Amplified products were detected by ethidium bromide staining of amplified products after agarose gel electrophoresis. When it was used to test 98 clinical specimens, this method was comparable to standard culture techniques in the detection, typing, and subtyping of influenza viruses.
Use of Presumpto Plates to identify anaerobic bacteria
Authors: Whaley D N , Wiggs L S , Miller P H , Srivastava P U , Miller J M
Published: January 2020

Identification of anaerobic bacteria requires special media and growth conditions that contribute to a higher cost per identification than that for aerobic isolates. Newer rapid methods streamline the identification process, but confirmation to the species level is often difficult. The Presumpto Plate method for the identification of commonly encountered anaerobes consists of three quadrant plates, each containing four conventional media, that result in the generation of 21 test parameters: growth on Lombard-Dowell medium; production of indole, indole derivative, catalase, lecithinase, and lipase; proteolysis of milk, H2S, and esculin; growth on 20% bile; precipitate on bile; DNase, glucose, casein, starch, and gelatin hydrolysis; and fermentation of lactose, mannitol, and rhamnose. Identification charts were developed by using the results from 2,300 anaerobic isolates. Because conventional media were used, there was a high degree of agreement between the Presumpto Plate method and the reference method when testing commonly encountered anaerobes. The Presumpto Plate method is as accurate as commercially available enzyme systems for the identification of many anaerobic species but is less expensive to perform.
Rapid susceptibility testing of Mycobacterium tuberculosis (H37Ra) by flow cytometry
Authors: Norden M A , Kurzynski T A , Bownds S E , Callister S M , Schell R F
Published: January 2020

The resurgence of tuberculosis has caused considerable effort to be focused on the development of rapid methods for determining the susceptibility of Mycobacterium tuberculosis to antimycobacterial agents. We demonstrated that susceptibility testing of M. tuberculosis can be accomplished rapidly by using flow cytometry. Results of tests were available within 24 h after M. tuberculosis organisms were incubated with ethambutol, isoniazid, rifampin, or streptomycin. The method was based on the ability of viable M. tuberculosis organisms to hydrolyze fluorescein diacetate (FDA) and the detection of fluorescent mycobacteria by flow cytometric analysis. The assay system also did not require multiplication of the mycobacteria. In contrast, M. tuberculosis organisms exposed to antimycobacterial agents hydrolyzed significantly less FDA. The use of flow cytometry and FDA staining shows considerable promise as a rapid method for obtaining susceptibility test results.
Evidence for the geographic spread of a methicillin-resistant Staphylococcus aureus clone between Portugal and Spain
Authors: Sanches I S , Ramirez M , Troni H , Abecassis M , Padua M , Tomasz A , de Lencastre H
Published: January 2020

Methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during a 7-month period in 1992 and 1993 at Hospital Pulido Valente (340 beds), Lisbon, Portugal, were characterized by a combination of genotypic and phenotypic methods. Clonal identities were determined by probing ClaI digests (i) with a mecA probe and (ii) with a Tn554 probe and (iii) by pulsed-field gel electrophoresis (PFGE) patterns of chromosomal SmaI digests. mecA-ClaI type I was predominant among these isolates (38 of 43). Most of these (37 of 38 [97.4%]) were associated with a single Tn554 pattern, pattern E, and the majority (23 of 38 [61%]) also showed a relatively uniform chromosomal background, as indicated by PFGE (PFGE pattern A). The major clone (mecA-ClaI type I::Tn554 type E and PFGE pattern A) at Hospital Pulido Valente was indistinguishable by these molecular typing criteria from the dominant clone that had been identified in two major current outbreaks of MRSA disease in Spain (Barcelona and Madrid). The Portuguese and Spanish clones also had a common heterogeneous class 3 phenotype and identical multidrug resistance patterns. The data presented in this work support the notion that MRSA clones can spread across considerable geographic distances.