bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis C virus (HCV) subtype prevalence in Chiang Mai, Thailand, and identification of novel subtypes of HCV major type 6
Authors: Doi H , Apichartpiyakul C , Ohba K I , Mizokami M , Hotta H
Published: January 2020

Subtype analysis of hepatitis C viruses (HCVs) obtained from patients with chronic liver disease in Chiang Mai, Thailand, was performed. Of 46 HCV isolates, 13 (28%) were shown to belong to HCV subtype 3a (HCV-3a), 10 (22%) to belong to HCV-1a, 7 (15%) to belong to HCV-1b, 1 (2%) to belong to HCV-3b, and 1 (2%) to belong to a variant group, as determined from partial nucleotide sequences of the NS5B region of the viral genome. Analysis of 5' untranslated region sequences identified five other isolates (11%) of HCV type 1 and two other isolates (4%) of type 3. Detailed phylogenetic positions for the variant described above and those previously obtained from blood donors and drug addicts in Chiang Mai were determined by a six-parameter neighbor-joining method on the basis of core, E1, and NS5B region sequences. The results revealed that those sequence variants represent novel subtypes of HCV type 6. The HCV type 6 isolates appear to be antigenically different from isolates of HCV types 1 and 2, as determined by a serotyping method that utilizes recombinant peptides corresponding to a portion of the NS4 protein. The significance of subtype analysis around this area is discussed.
Rapid identification of Candida species by DNA fingerprinting with PCR
Authors: Thanos M , Schonian G , Meyer W , Schweynoch C , Graser Y , Mitchell T G , Presber W , Tietz H J
Published: January 2020

DNA polymorphisms in different species and strains of the genus Candida were assessed by amplifying genomic DNA with single nonspecific primers. This PCR method employed an arbitrary primer (the 10-mer AP3), a primer derived from the intergenic spacer regions (T3B), and the microsatellite primers (GTG)5 and (AC)10. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The numbers and sizes of the amplification products were characteristic for each species. All yeast species tested could be clearly distinguished by their amplification patterns. With all primers, PCR fingerprints also displayed intraspecies variability. However, PCR profiles obtained from different strains of the same species were far more similar than those derived from different Candida species. By comparing species-specific PCR fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by routine biochemical methods.
Characteristics of Escherichia coli strains belonging to enteropathogenic E. coli serogroups isolated in Italy from children with diarrhea
Authors: Giammanco A , Maggio M , Giammanco G , Morelli R , Minelli F , Scheutz F , Caprioli A
Published: January 2020

Fifty-five Escherichia coli strains belonging to enteropathogenic E. coli (EPEC) serogroups were examined for phenotypic and genetic factors associated with virulence. The strains were isolated in Italy from children with diarrhea and identified as EPEC by clinical laboratories using commercially available antisera. O:H serotyping showed that 35 strains (27 of O26, O111, and O128 serogroups) belonged to 11 serotypes considered to be classical EPEC O:H serotypes. The other 20 isolates were classified as 15 nonclassical EPEC O:H serotypes. All the potential EPEC virulence factors associated with bacterial adhesion (localized adherence, fluorescentactin staining test positivity, presence of the attaching and effacing [eaeA] gene), the production of verotoxin, and the positivity with the enterohemorrhagic E. coli probe were significantly more frequent among isolates belonging to classical than nonclassical serotypes. Strains displaying an aggregative adhesion and hybridizing with the enteroaggregative DNA probe were found in serogroups O86, O111, and O126. Verotoxin-producing isolates belonged to serogroups O26, O111, and O128. Only one of the isolates hybridized with the EPEC adherence factor (EAF) probe, but 33 strains gave positive results with the eae probe, confirming that the former is more suitable in epidemiological studies in European countries. These results indicate that up to 75% of strains identified as EPEC by commercial antisera may possess potential virulence properties and/or belong to classical EPEC O:H serotypes and suggest that O grouping is still a useful diagnostic tool for presumptive identification of diarrheagenic E. coli in clinical laboratories.
Immunoserologic evidence of coinfection with Borrelia burgdorferi, Babesia microti, and human granulocytic Ehrlichia species in residents of Wisconsin and Minnesota
Authors: Mitchell P D , Reed K D , Hofkes J M
Published: January 2020

In Wisconsin and Minnesota, Ixodes scapularis (Ixodes dammini) ticks are the vector of three microorganisms that may cause significant disease in humans and lower mammals. These diseases include Lyme borreliosis, which is caused by Borrelia burgdorferi, babesiosis, which is caused by Babesia microti, and human granulocytic ehrlichiosis (HGE), which is caused by an apparently new species in the genus Ehrlichia. Immunoserologic testing was performed on sera from patients with a diagnosis of one of these diseases to determine if there was evidence of coinfection with one or more of the other agents. Of 96 patients with Lyme borreliosis, 9 (9.4%) demonstrated immunoserologic evidence of coinfection: 5 (5.2%) with the agent of HGE, 2 (2.1%) with B. microti, and 2 (2.1%) with both microorganisms. Of 19 patients diagnosed with HGE, 3 (15.8%) showed immunoserologic evidence of coinfection: 1 (5.3%) with B. burgdorferi, 1 (5.3%) with B. microti, and 1 (5.3%) with both microorganisms. One patient diagnosed with babesiosis was also seropositive for ehrlichiosis. These results provide evidence for coinfection, perhaps explaining the variable manifestations and clinical responses noted in some patients with tick-transmitted diseases. In certain clinical settings, laboratory testing for coinfection is indicated to ensure that appropriate antimicrobial treatment is given.
Identification of intermediately virulent Rhodococcus equi isolates from pigs
Authors: Takai S , Fukunaga N , Ochiai S , Imai Y , Sasaki Y , Tsubaki S , Sekizaki T
Published: January 2020

We recently reported the existence of Rhodococcus equi isolates with at least three virulence levels, isolated from AIDS patients: virulent R. equi having 15- to 17-kDa antigens that kills mice with 10(6) cells, intermediately virulent R. equi having a 20-kDa antigen that kills mice with 10(7) cells, and avirulent R. equi that does not kill mice with 10(8) cells or more (S. Takai, Y. Imai, N. Fukunaga, Y. Uchida, K. Kamisawa, Y. Sasaki, S. Tsubaki, and T. Sekizaki, J. Infect. Dis. 172:1306-1311, 1995). Virulent R. equi having the 15- to 17-kDa antigens has been isolated frequently from horses and their environment, but the source of intermediately virulent R. equi having the 20-kDa antigen is poorly understood. There are many reports of the isolation of R. equi from the lymph nodes of pigs with and without lesions resembling those of tuberculosis. Therefore, we analyzed antigens of R. equi isolates from the submaxillary lymph nodes of pigs by immunoblotting with monoclonal antibodies against these virulence-associated antigens. Immunoblots of whole-cell antigen preparations of R. equi pig isolates revealed the presence of the 20-kDa antigen in almost all the pig isolates studied, and these isolates were intermediately virulent for mice. We also demonstrated that the expression of the 20-kDa antigen and its pathogenicity in mice were associated strongly with the presence of five large, distinct plasmids of 70 to 95 kb; two of the five plasmids from pig isolates were the same sizes as those from human isolates. These results suggest that R. equi having the 20-kDa antigen exists in the submaxillary lymph nodes of pigs and that the source of infection in some human cases might be associated with pigs and their environment.
Most frequent scenario for recurrent Candida vaginitis is strain maintenance with "substrain shuffling": demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2
Authors: Lockhart S R , Reed B D , Pierson C L , Soll D R
Published: January 2020

The following three basic scenarios have emerged for the genetic relatedness of strains in recurrent vaginal candidiasis: strain maintenance without genetic variation, strain maintenance with minor genetic variation, and strain replacement. To test the frequency of each of the three scenarios, the genetic relatedness of Candida albicans isolates from each of 18 patients with recurrent infections was assessed by sequential DNA fingerprinting with the following three probes: the Ca3 probe; the C1 probe, a subfragment of the Ca3 probe which hybridizes to hypervariable genomic fragments; and the unrelated CARE2 probe. In each of the 18 patients with recurrent infections, the same strain was responsible for sequential infections, suggesting that the predominant scenario is strain maintenance. However, in 56% of these patients, the strain exhibited minor genetic variations in sequential infections. These changes were not found to be progressive. Rather, the changes suggest that substrains of an established infecting strain are shuffled in sequential infections. Results are also presented that in 45% of patients with recurrent infections, oral and vulvovaginal isolates were identical, in 35% they were highly related but not identical, and in 20% they were unrelated. These results differ markedly from those for commensal isolates simultaneously cultured from the oral cavity and vulvovaginal region of healthy individuals. Finally, it is demonstrated that in all eight cases in which C. albicans was isolated from both the male sexual partner of the patient with a recurrent infection and the patient, an isolate from the male partner was identical or highly related to the vulvovaginal strain. These results demonstrate that in patients with recurrent vulvovaginitis, a single strain usually dominates both in the different body locations of the patient and in the male partner and that it is maintained through sequential infections. However, in patients with recurrent infections, different substrains of the established clone dominate in an apparently random fashion, a process that we refer to as "substrain shuffling".
Diagnosis of contagious caprine pleuropneumonia by detection and identification of Mycoplasma capricolum subsp. capripneumoniae by PCR and restriction enzyme analysis
Authors: Bölske G , Mattsson J G , Bascuñana C R , Bergström K , Wesonga H , Johansson K E
Published: January 2020

Contagious caprine pleuropneumonia (CCPP), one of the most serious and dramatic diseases of goats, is caused by Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae). This organism is very difficult to isolate and to correctly identify. In a previous report we described a method for the rapid detection and identification of M. capripneumoniae. This method is based on a PCR system by which a segment of the 16S rRNA gene from all mycoplasmas of the M. mycoides cluster can be amplified. The PCR product is then analyzed by restriction enzyme cleavage for the identification of M. capripneumoniae DNA. This system has now been further evaluated with respect to specificity and diagnostic efficacy for the identification and direct detection of the organism in clinical material. Identification by restriction enzyme analysis of amplified DNA from mycoplasmas of the M. mycoides cluster was verified for 55 strains, among which were 15 strains of M. capripneumoniae. The PCR was applied to clinical samples from the nose, ear, pharynx, pleural fluid, and lung tissue containing M. capripneumoniae or other mycoplasmas. As expected, mycoplasmas belonging to the M. mycoides cluster could be detected by the PCR. Restriction enzyme analysis of the PCR products could then be applied for the identification of M. capripneumoniae. Clinical samples and cultures containing M. capripneumoniae were dried on filter paper, to try an easier sample transport method, and were tested by PCR. M. capripneumoniae DNA could be detected in the dried specimens, but the sensitivity of the PCR test was reduced.
Human papillomavirus type 70 genome cloned from overlapping PCR products: complete nucleotide sequence and genomic organization
Authors: Forslund O , Hansson B G
Published: January 2020

The genome of human papillomavirus (HPV) type 70 (HPV 70), isolated from a cervical condyloma, was obtained by cloning overlapping PCR products. By automated DNA sequence analysis, the genome was found to consist of 7,905 bp with a G + C content of 40%. The genomic organization showed the characteristic features shared by other sequenced HPVs. Nucleotide sequence comparison with previously known HPV types demonstrated the closest homology with HPV 68 (82%), HPV 39 (82%), HPV 18 (70%), HPV 45 (70%), and HPV 59 (70%). Comparison with seven other partially sequenced HPV 70 isolates showed homologies of between 100 and 99.5%. Cloning of overlapping PCR products and automated DNA sequence analysis was found to be a feasible method of obtaining full-length sequences of HPVs.
Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and antimicrobial resistance patterns
Authors: Hall L M , Whiley R A , Duke B , George R C , Efstratiou A
Published: January 2020

A collection of 54 isolates of invasive Streptococcus pneumoniae of serotypes 3 and 14 and serogroups 6, 9, 19, and 23 was investigated. Multilocus enzyme electrophoresis and pulsed-field gel electrophoresis suggested that two clones were represented in the collection, one of serotype 14 isolates, most of which were resistant to erythromycin, and one of serotype 9V isolates, in which resistance to penicillin (MIC, 1 microgram/ml), cefotaxime, and co-trimoxazole was common. Among other isolates there were only a limited correlation between genetic relatedness measured by multilocus enzyme electrophoresis and expression of the same capsule type. However, isolates with highly related pulsed-field gel electrophoresis patterns always shared the same serotype and highly related allele profiles. Calculation of the index of association suggests a freely recombining population structure with epidemic spread of successful clones.
Coinfection with human herpesvirus 6 variants A and B in lung tissue
Authors: Cone R W , Huang M L , Hackman R C , Corey L
Published: January 2020

Human herpesvirus 6 (HHV-6) variant B is frequently identified in peripheral blood, but identification of HHV-6 variant A is relatively rare. We devised a PCR-based method for sensitive, simultaneous detection of both HHV-6 variants. The method was applied to 34 lung tissue specimens that were previously shown to contain HHV-6 DNA. A total of 22 lung tissue samples showed coinfections with HHV-6 variants A and B, 2 had only HHV-6 variant A DNA, and 10 had only HHV-6 variant B DNA. The prevalences of coinfections in lung tissues from healthy controls (54% coinfected) and in those from bone marrow transplant patients with pneumonia (67% coinfected) were similar. These data indicate that coinfections of HHV-6 variants A and B commonly occur in lung tissues of healthy and diseased individuals.
PCR for detection of Clostridium botulinum type C in avian and environmental samples
Authors: Franciosa G , Fenicia L , Caldiani C , Aureli P
Published: January 2020

A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak.
Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR
Authors: Cogswell F B , Bantar C E , Hughes T G , Gu Y , Philipp M T
Published: January 2020

It is demonstrated that a diagnostic PCR for Borrelia burgdorferi can be inhibited in the presence of more than 500 ng of host (monkey skin) DNA. The inhibitor is the host DNA itself. An acceptable value for analytical sensitivity can be obtained by diluting the skin-B. burgdorferi proteinase K lysate to a level below the inhibitory concentration of the host DNA. Dilution of the lysate may obviate the need for further DNA purification.
Studies of in vivo distribution of bovine herpesvirus type 4 in the natural host
Authors: Egyed L , Ballagi-Pordány A , Bartha A , Belák S
Published: January 2020

The in vivo distribution of bovine herpesvirus type 4 (BHV-4) was examined by testing nasal and conjunctival exudates, peripheral blood leukocytes, and various organs of experimentally infected calves. For virus detection, a nested PCR assay, virus isolation, and immunohistochemistry were applied. The nervous system and the muscles were free of viral DNA. Liver and intestinal lymph nodes contained low amounts of virus (less than two copies per 1 microgram of cellular DNA). Intestinal, tonsil, thymus, and kidney tissues contained more viral DNA copies (5 to 50 copies per 1 microgram of cellular DNA). The highest amounts of BHV-4 DNA (50 to 500 copies per 1 microgram of cellular DNA) were found in the spleen, lungs, trachea, and nasal epithelium. Amplification of DNA from blood lymphocytes through postinoculation (p.i.) day 48 proved that the virus started to replicate in these cells immediately after inoculation of the calves and that intensive virus growth took place during the 7 to 8 weeks of the infection. The number of virus-infected lymphocytes reached the maximum on p.i. days 22 to 26 and slowly declined thereafter. Virus-infected cells were found only in the spleen on p.i. day 48 by immunohistochemistry. Western blotting (immunoblotting) detected signs of an immune response against 9 of the 29 BHV-4 proteins.
Comparison of PCR-based approaches to molecular epidemiologic analysis of Clostridium difficile
Authors: Collier M C , Stock F , DeGirolami P C , Samore M H , Cartwright C P
Published: January 2020

Representative isolates of the 10 serogroups of Clostridium difficile and 39 clinical isolates (30 toxigenic and 9 nontoxigenic), including 5 isolates from a confirmed nosocomial outbreak, were analyzed by using two previously described arbitrary-primer PCR (AP-PCR) molecular typing methodologies (AP-PG05 and AP-ARB11) and PCR ribotyping. The two AP-PCR methods investigated gave comparable results; AP-PG05 and AP-ARB11 identified 8 and 7 groups among the serogroup isolates and classified the clinical isolates into 21 and 20 distinct groups, respectively. PCR ribotyping also identified 8 unique groups among the serogroup isolates but classified the clinical isolates into 23 groups. In addition, when results obtained by the PCR methods were compared with typing data generated by pulsed-field gel electrophoresis (PFGE), PCR ribotyping and PFGE were found to be in agreement for 83% (29 of 35) of isolates typeable by both techniques while AP-PG05 was in agreement with PFGE for 60% (20 of 33) and AP-ARB11 was in agreement with PFGE for only 44% (17 of 36). These results indicate that PCR ribotyping is a more discriminatory approach than AP-PCR for typing C. difficile and, furthermore, that this technique generates results that are in higher concordance with those obtained by using an established method for differentiating isolates of this organism on a molecular level than are results generated by using AP-PCR.
Identification of bovine Neospora parasites by PCR amplification and specific small-subunit rRNA sequence probe hybridization
Authors: Ho M S , Barr B C , Marsh A E , Anderson M L , Rowe J D , Tarantal A F , Hendrickx A G , Sverlow K , Dubey J P , Conrad P A
Published: January 2020

Neospora is a newly recognized genus of pathogenic coccidia, closely related to Toxoplasma gondii, that can cause abortion or congenital disease in a variety of domestic animal hosts. On the basis of the small-subunit rRNA gene sequences of Neospora spp. and other apicomplexa coccidia, oligonucleotide primers COC-1 and COC-2 were used for PCR amplification of conserved sequences of approximately 300 bp in size. A Neospora-specific chemiluminescent probe hybridized to Southern blots of amplification products from Neospora DNA but not to Southern blots with amplified DNA from the other coccidian parasites tested. A Toxoplasma-specific probe whose sequence differed from that of the probe for Neospora spp. by a single base pair was used to distinguish these parasites by specific Southern blot hybridization. The PCR system detected as few as one Neospora tachyzoite in the culture medium or five tachyzoites in samples of whole blood or amniotic fluid spiked with Neospora parasites. In addition, Neospora PCR products were successfully amplified from whole blood and amniotic fluid samples of experimentally infected bovine and rhesus macaque fetuses. These results indicate that this PCR and probe hybridization system could be a valuable adjunct to serology and immunohistochemistry for the diagnosis of Neospora infections in bovine or primate fetuses.
Identification and differentiation of Mycobacterium avium and M. intracellulare by PCR
Authors: Chen Z H , Butler W R , Baumstark B R , Ahearn D G
Published: January 2020

Known DNA sequences coding for the 16S rRNAs of 14 slowly growing Mycobacterium species were analyzed. Three sets of primers were synthesized: MAV and MIN, for M. avium and M. intracellulare, respectively, and MYCOB, for the slowly growing mycobacteria. Whole-cell DNAs of 14 reference species were extracted and amplified by PCR with the MYCOB, MAV, and MIN primers. The MYCOB primer amplified a 0.9-kb segment from the DNAs of all 14 species. The MAV and MIN primers each amplified one highly specific 1.3-kb segment from the homologous DNA, respectively. DNAs from each of 10 clinical isolates of M. avium and M. intracellulare identified by conventional methods were amplified with the MYCOB as well as the MAV and MIN primers; 9 of 10 isolates of each species were identified with their respective primers. One isolate of M. intracellulare was subsequently found to have been mislabeled. One isolate designated M. avium reacted only with the MYCOB primer. The hypervariable region of this strain was shown by DNA sequence analysis to be distinct from all known 16S rRNA sequences of Mycobacterium spp. Our data indicate that the currently identified M. avium-M. intracellulare complex includes strains genetically diverse from M. avium and M. intracellulare.
Bovine adenovirus type 10 identified in fatal cases of adenovirus-associated enteric disease in cattle by in situ hybridization
Authors: Smyth J A , Benkö M , Moffett D A , Harrach B
Published: January 2020

A severe, naturally occurring enteric disease of cattle in which adenovirus inclusions are present in the intestinal vascular endothelium has been recognized in several countries; three different adenovirus serotypes have been isolated from affected animals. An in situ hybridization technique for the detection of bovine adenoviral DNA was developed and applied to tissue from 13 cattle in Northern Ireland diagnosed to have the disease. Bovine adenovirus serotype 10 (BAV-10) was identified in the vascular inclusions of all cattle, providing strong evidence that adenoviral enteric vascular disease in cattle is associated with this serotype. The existence of BAV-10 has only recently been recognized. The first molecular biology-based technique for the diagnosis of BAV-10 infection is described. The animals in the present study are the first in which BAV-10 has had a confirmed role in a pathologic process.
Diagnosis by AMPLICOR PCR of Chlamydia trachomatis infection in urine samples from women and men attending sexually transmitted disease clinics
Authors: Quinn T C , Welsh L , Lentz A , Crotchfelt K , Zenilman J , Newhall J , Gaydos C
Published: January 2020

Screening of urine specimens from men for Chlamydia trachomatis infection by a commercial PCR assay (AMPLICOR C. trachomatis Test; Roche Diagnostic Systems, Inc., Branchburg, N.J.) is a sensitive and specific noninvasive diagnostic assay. Since screening of women for C. trachomatis infection with the AMPLICOR C. trachomatis Test has been limited to use with endocervical swab specimens, we conducted an evaluation of the AMPLICOR C. trachomatis Test for the detection of C. trachomatis using female urine samples and compared the results of those obtained by in vitro culture and PCR of endocervical swab specimens. For 713 men we compared the performance of AMPLICOR C. trachomatis Test with urine specimens with that of culture of urethral specimens. For specimens that were PCR positive and culture negative, two additional tests were used to resolve the discrepancies: direct fluorescent-antibody assay (DFA) of sediment from a spun endocervical specimen culture vial and major outer membrane protein-based PCR of the sediment from the endocervical specimen culture vial. Of 525 urine specimens from females, 67 (12.8%) were PCR positive, and 41 (7.8%) endocervical specimens from the 525 women were culture positive. After resolution of the discrepancies, the resolved sensitivity of the urine PCR was 93.3%, whereas the sensitivity of endocervical swab specimen culture was 67.3%. Of 468 female endocervical swab specimens, 47 (10.0%) had a positive PCR result and 33 (7.0%) were culture positive. The resolved sensitivity of the endocervical swab specimen PCR was 86%. Of 415 matched female urine and endocervical swab specimens, there were 49 confirmed infections; 30 (61.2%) specimens were positive by culture of the endocervical swab specimen, 40 (81.6%) were positive by confirmed endocervical swab specimen PCR, 43 (87.8%) were positive by confirmed urine PCR, and all 49 (100%) were positive by either endocervical swab specimen PCR or urine PCR. For men, the resolved sensitivity of the urine PCR was 88%, and the sensitivity of culture was only 50.7%. These results indicate that urine PCR is highly sensitive for the detection of C. trachomatis in both women and men and provides a noninvasive technique for routine screening for chlamydial infection.
Utility of PCR in diagnosing pulmonary tuberculosis
Authors: Bennedsen J , Thomsen V O , Pfyffer G E , Funke G , Feldmann K , Beneke A , Jenkins P A , Hegginbothom M , Fahr A , Hengstler M , Cleator G , Klapper P , Wilkins E G
Published: January 2020

At present, the rapid diagnosis of pulmonary tuberculosis rests with microscopy. However, this technique is insensitive and many cases of pulmonary tuberculosis cannot be initially confirmed. Nucleic acid amplification techniques are extremely sensitive, but when they are applied to tuberculosis diagnosis, they have given variable results. Investigators at six centers in Europe compared a standardized PCR system (Amplicor; Roche) against conventional culture methods. Defined clinical information was collected. Discrepant samples were retested, and inhibition assays and backup amplification with a separate primer pair were performed. Mycobacterium tuberculosis complex organisms were recovered from 654 (9.1%) of 7,194 samples and 293 (7.8%) of 3,738 patients. Four hundred fifty-two of the M. tuberculosis isolates from 204 patients were smear positive and culture positive. Among the culture-positive specimens, PCR had a sensitivity of 91.4% for smear-positive specimens and 60.9% for smear-negative specimens, with a specificity of 96.1%. Analysis of 254 PCR-positive, culture-negative specimens with discrepant results revealed that 130 were from patients with recently diagnosed tuberculosis and 94 represented a presumed laboratory error. Similar analysis of 118 PCR-negative, culture-positive specimens demonstrated that 27 discrepancies were due to presumed uneven aliquot distribution and 11 were due to presumed laboratory error; PCR inhibitors were detected in 8 specimens. Amplicor enables laboratories with little previous experience with nucleic acid amplification to perform PCR. Disease in more than 60% of the patients with tuberculosis with smear-negative, culture-positive specimens can be diagnosed at the time of admission, and potentially all patients with smear-positive specimens can immediately be confirmed as being infected with M. tuberculosis, leading to improved clinical management.
Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR
Authors: Langemeier J L , Cook S J , Cook R F , Rushlow K E , Montelaro R C , Issel C J
Published: January 2020

Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detected in 63 of 63 horses with EIAV antibody test-positive histories on approved serologic tests, demonstrating that RT-nPCR was probably directed against highly conserved sequences in the viral genome. The RT-nPCR assay, agar gel immunodiffusion test, and conventional virus isolation were compared for detection of early infection in 12 experimentally infected ponies. Viral gag sequences were detected in all 12 animals by 3 days postinfection (p.i.) by RT-nPCR, whereas virus could not be routinely isolated on cell culture until 9 to 13 days p.i. and EIAV antibodies could not be detected by agar gel immunodiffusion until 20 to 23 days p.i. Finally, specificity of the RT-nPCR assay was examined by testing plasma from 43 horses with serologic test-negative histories and no known contact with EIAV-infected animals. Viral gag sequences were not detectable in this control group. These data suggest that the EIAV RT-nPCR assay effectively detects EIAV and is more sensitive than current standard methods for detection of early stages of infection.
Diagnostic implications of kinetics of immunoglobulin M and A antibody responses to Toxoplasma gondii
Authors: Gorgievski-Hrisoho M , Germann D , Matter L
Published: January 2020

We evaluated immunoglobulin M (IgM) and IgA assays that could improve the predictive value for recently acquired toxoplasma infection for patients with positive screening test results. Follow-up sera were collected from 82 patients whose initial serum specimen had a reactive anti-Toxoplasma gondii IgM result. According to the evolution of the immune response, patients were divided retrospectively into two groups: one in which a recent infection was unlikely and the other one with an evolving immune response suggestive of recent toxoplasma infection. All IgM and one of three IgA assays used in the study are suitable for screening pregnant patients, with a negative predictive value of 100%. The predictive value of positive results is much lower because of the low prevalence of acute toxoplasmosis in pregnant women and the long persistence of IgM after acute infection. In the present study, all except one IgM enzyme immunoassay remained positive well beyond 6 months after the initial sample was tested. The IgM immunofluorescence test had the shortest persistence of positivity in most cases. IgA tests were either too insensitive or remained reactive too long to be useful for screening pregnant patients. Interpreting enzyme immunoassays with modified cutoff values and the combination of two tests could improve the predictive value of positive results to about 80% in terms of recent infection.
Duration of incubation of fungal cultures
Authors: Morris A J , Byrne T C , Madden J F , Reller L B
Published: January 2020

To determine the optimum duration of incubation for recovery of fungi, the results of 2,173 consecutive clinical cultures were reviewed. Overall, 94% of fungal isolates were detected by day 7 and 98% were detected by day 14. Yeasts were usually (98%) detected within the first week of incubation. Recovery of molds required more time, but 81% were detected by day 7 and more than 96% were detected by day 14.
PCR of peripheral blood for diagnosis of meningococcal disease
Authors: Newcombe J , Cartwright K , Palmer W H , McFadden J
Published: January 2020

Meningococcal disease is normally suspected on clinical grounds and is confirmed by isolation of Neisseria meningitidis bacteria from blood or cerebrospinal fluid or, more recently, by serology or PCR of cerebrospinal fluid. Achieving confirmation of a clinical diagnosis of meningococcal disease has become more difficult in the last few years. The pre-hospitalization administration of parenteral benzylpenicillin normally renders blood cultures sterile, and lumbar puncture is undertaken less frequently, especially in young children. We evaluated PCR for the detection of meningococcal DNA in 80 blood samples taken from patients with known or suspected meningococcal disease or from patients with other diagnoses (negative controls). Both the sensitivity and the specificity of the test were 100% for patients with confirmed cases of meningococcal disease when the blood buffy coat was used (83 to 100% sensitivity and 87 to 100% specificity with 95% confidence limits). Positive PCR results could be obtained from both blood buffy coat and serum samples. Sensitivity was unaffected by prior antibiotic treatment. PCR is a rapid, sensitive test that may be used to confirm a diagnosis of meningococcal disease by using peripheral blood samples. Introduction of this test into clinical laboratories may in some cases obviate the need for lumbar puncture to be performed on patients with suspected meningococcal disease. Our results demonstrate that a substantial number of cases of meningococcal disease are not confirmed by conventional techniques and remain undiagnosed. If the PCR test described here was widely applied, the number of cases of meningococcal disease ascertained might rise by as much as 60% greater than that recognized at present. It is likely that we are in a prevaccination era for meningococcal disease. Better case ascertainment is urgently required to assess the need for vaccines, to determine their costs and benefits, and to monitor their efficacies.
DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora
Authors: Wakefield A E
Published: January 2020

Samples of ambient air collected with three different types of spore traps in a rural location were examined for the presence of Pneumocystis carinii by screening for P. carinii-specific DNA sequences by DNA amplification. Eleven spore trap samples were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of P. carinii f. sp. carinii and P. carinii f. sp. hominis. The samples were collected over a 3-year period during the months of May to September, with a range of sampling times from 9 to 240 h. One air sample from an animal facility housing P. carinii-infected rats was also examined. P. carinii-specific amplification products were obtained from samples from each of the spore traps. The amplification products from eight air samples were cloned and sequenced. The majority of the recombinants from each of these samples had sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis, and a number of clones had single-base differences. These data suggest that sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis can be detected in samples of air collected in a rural location and that P. carinii may be a component of the air spora of rural Oxfordshire.
Revised approach for identification and detection of ampicillin and vancomycin resistance in Enterococcus species by using MicroScan panels
Authors: Iwen P C , Kelly D M , Linder J , Hinrichs S H
Published: January 2020

The frequency of antimicrobial agent-resistant enterococci is increasing, making accurate identification and screening for susceptibility essential. We evaluated the ability of MicroScan Positive Breakpoint Combo Type 6 panels (Dade MicroScan Inc., West Sacramento, Calif.) to identify Enterococcus species and to detect ampicillin and vancomycin resistance. A total of 398 well-characterized Enterococcus isolates from two institutions were inoculated into MicroScan panels, into conventional biochemical assays, and into ampicillin and vancomycin agar dilution media. Resistance was verified by the broth macrodilution method. MicroScan panels accurately detected resistance to ampicillin in 132 of 132 enterococcal isolates, while three isolates for which the MICs were < 16 micrograms/ml were classified incorrectly by MicroScan panels as resistant. No beta-lactamase-producing enterococci were detected. All 64 isolates showing resistance to vancomycin (MICs > or = 32 micrograms/ml) were correctly classified by MicroScan panels. Seven isolates for which the vancomycin MICs were 8 and 16 micrograms/ml were incorrectly classified as susceptible by MicroScan panels, while eight isolates for which the MICs were 4 micrograms/ml were incorrectly labeled as intermediate. Fourteen of these 15 isolates were subsequently identified as motile enterococci. Overall, there were three major errors in susceptibility testing for ampicillin and 15 minor errors for vancomycin. Conventional testing confirmed the identity of 181 Enterococcus faecalis isolates, 157 E. faecium isolates, and 60 isolates of other species; however, 56 of these 60 isolates were misidentified by the MicroScan panels. After recognition of this problem, a revised approach which included tests for pigment, motility, and sucrose fermentation was devised. In combination with these additional assays, the conventional MicroScan panels accurately identified the 56 originally misidentified isolates. In summary, the ability of MicroScan panels to detect vancomycin and ampicillin resistance in enterococci was confirmed. Our study found that the inability of MicroScan panels to identify enterococci other than E. faecalis and E. faecium can be compensated for by the addition of standard assays.
Precore mutant of hepatitis B virus prevails in acute and chronic infections in an area in which hepatitis B is endemic
Authors: Chu C M , Yeh C T , Chiu C T , Sheen I S , Liaw Y F
Published: January 2020

By using an amplification-created restriction site method, the precore TAG mutant of hepatitis B virus was detected in 6 (75%) of 8 acute fulminant hepatitis B patients, 7 (58%) of 12 acute self-limiting hepatitis B patients, 35 (81%) of 43 hepatitis B virus surface antigen carriers with fulminant hepatitis, and 42 (70%) of 60 hepatitis B virus surface antigen carriers with chronic hepatitis. The precore TAG mutant prevails in acute and chronic hepatitis B of various severity in this area where hepatitis B is endemic.
Clonal diversity of oral Eikenella corrodens within individual subjects by arbitrarily primed PCR
Authors: Chen C , Ashimoto A
Published: January 2020

The genetic diversity of 205 Eikenella corrodens isolates recovered from dental plaque, mucosal surfaces, and saliva of 24 subjects was examined by arbitrarily primed PCR. Twenty-two subjects were colonized by multiple clones (range, two to eight; mean, 3.7). This study demonstrates the utility of arbitrarily primed PCR for clonal analysis of E. corrodens and the multiclonal colonization of E. corrodens in the oral cavity.
Mycobacterium genavense infections in pet animals
Authors: Kiehn T E , Hoefer H , Bottger E C , Ross R , Wong M , Edwards F , Antinoff N , Armstrong D
Published: January 2020

Mycobacterium genavense, a recently reported cause of a wasting illness in patients with AIDS, was isolated from a cervical lymph mode from a dog with severe hind limb weakness and from trachael tissue from a parrot with acute onset respiratory distress. Physicians caring for immunocompromised patients should consider birds and dogs potential sources of M. genavense infection and submit appropriate specimens for culture.
Auxotypes and serogroups of tetracycline-resistant Neisseria gonorrhoeae isolated in Malaysia
Authors: Koay A S , My R , Cheong Y M
Published: January 2020

Between 1992 and 1994, 253 tetracycline-resistant Neisseria gonorrhoeae (TRNG) strains were isolated and characterized by auxotype and serogroup (A/S) classes to study TRNG prevalence in different years. TRNG accounted for 28.1, 42.5, and 51.9% of the strains isolated in 1992, 1993, and 1994, respectively, showing a significant increase in each successive year (chi square = 26.7, P < 0.001). There was no significant increase in penicillinase-producing TRNG, which accounted for 53.1, 53.8, and 63.2% of the TRNG isolates. The 253 TRNG isolates belonged to 53 A/S classes. Eighteen A/S classes not observed in 1992 were detected in 1993, and 11 A/S classes not observed in 1992 and 1993 were isolated in 1994, indicating dissemination of the tetracycline resistance gene among the N. gonorrhoeae strains in Malaysia. Its emergence and subsequent rapid spread are alarming. The plasmid is capable of self-transfer (S.A. Morse, S.R. Johnson, J.W. Biddle, and M.C. Roberts, J. Infect. Dis. 155:819-822, 1987), allowing further dissemination of tetracycline resistance.
Comparison of Isolator 1.5 and BACTEC NR660 aerobic 6A blood culture systems for detection of fungemia in children
Authors: Petti C A , Zaidi A K , Mirrett S , Reller L B
Published: January 2020

The Isolator 1.5 microbial system (ISO 1.5) (Wampole Laboratories, Cranbury, N.J.) was compared with the BACTEC NR660 aerobic NR6A bottle (NR6A) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of fungemia in hospitalized pediatric patients. For 4,825 paired blood cultures evaluated retrospectively from April 1992 to December 1994, at least one blood culture system was positive for 89 clinically important fungal isolates involved in 36 episodes of fungemia in 34 patients. Sixty isolates (44 Candida albicans, 12 Candida parapsilosis, and 4 Candida tropicalis isolates) were recovered from both systems, 13 were recovered from NR6A bottles only (10 C. albicans, 1 C. parapsilosis, and 2 Cryptococcus neoformans isolates), and 16 were recovered from ISO 1.5 tubes only (8 C. albicans and 5 C. parapsilosis isolates and 1 C. tropicalis, 1 Candida lusitaniae, and 1 Rhodotorula glutinis isolate) (P > 0.05). For the 60 Candida isolates from both systems, the mean time to detection was the same in each system. Thirty-seven isolates were detected by both systems on the same day, 9 isolates were detected earlier by NR6A, and 14 isolates were detected earlier by ISO 1.5 (P > 0.05). Of the 36 clinically important episodes of fungemia, 22 were detected by both systems (13 C. albicans isolates and 9 other Candida isolates), 4 were detected by NR6A only (3 C. albicans isolates and 1 C. neoformans isolate), and 10 were detected by ISO 1.5 only (3 C. albicans isolates, 6 other Candida isolates, and 1 R. glutinis isolate) (P > 0.05). Of the 22 episodes in which cultures from both systems were positive at some point during the episode, 12 were detected on the same day by both systems, 8 were detected earlier by NR6A, and 2 were detected earlier by ISO 1.5. Thus, for our pediatric population, NR6A is comparable to ISO 1.5 in both yield and time to detection of yeasts in fungemic patients.
Rapid detection of Mycobacterium avium in stool samples from AIDS patients by immunomagnetic PCR
Authors: Li Z , Bai G H , von Reyn C F , Marino P , Brennan M J , Gine N , Morris S L
Published: January 2020

Direct PCR detection of bacteria in clinical samples is often hindered by the presence of compounds that inhibit the PCR. To improve and accelerate the diagnosis of Mycobacterium avium-M. intracellulare complex infections, an immunomagnetic PCR (IM-PCR) assay was developed. This IM-PCR procedure combines the separation of mycobacteria by antimycobacterial monoclonal antibody coupled to magnetic beads with an M. avium-M. intracellulare complex-specific PCR protocol based on 16S rRNA gene sequences. As few as 10 M. avium bacilli were detected in spiked human stool samples, a clinical specimen usually refractory to conventional PCR analysis, by the IM-PCR method. Moreover, M. avium organisms were detected in about 24 h in 18 of 22 culture-confirmed fecal samples from AIDS patients. This IM-PCR protocol should allow for the rapid and sensitive detection of M. avium isolates in clinical specimens.
Enolase antigen, mannan antigen, Cand-Tec antigen, and beta-glucan in patients with candidemia
Authors: Mitsutake K , Miyazaki T , Tashiro T , Yamamoto Y , Kakeya H , Otsubo T , Kawamura S , Hossain M A , Noda T , Hirakata Y , Kohno S
Published: January 2020

We compared the specificities and sensitivities of four tests used for the serodiagnosis of candidemia in 39 patients with candidemia, including 10 patients with superficial Candida colonization, 10 patients with deep mycosis, and 20 healthy subjects. The results obtained by the dot immunoblotting assay for detecting the enolase antigen (48 kDa) were compared with those of assays for detecting mannan antigen, heat-labile antigen (a threshold titer of four times), and beta-glucan (> or = 60 pg/ml). Enolase antigen was detected in 28 (71.8%) patients with candidemia, while 30 (76.9%), 10 (25.6%), and 27 (84.4%) patients were positive for the heat-labile antigen by the Cand-Tec assay, the mannan antigen by the Pastorex Candida assay, and beta-glucan by the limulus test, respectively. Ten patients with superficial Candida colonization, 5 patients with invasive pulmonary aspergillosis, 5 patients with cryptococcosis, and 20 healthy subjects were negative for both enolase antigen and mannan antigen. Two patients with superficial Candida colonization, one patient with invasive pulmonary aspergillosis, and two patients with cryptococcosis were positive by the Cand-Tec assay. The beta-glucan concentration was more than 60 pg/ml in all patients with invasive pulmonary aspergillosis; however, it was less than 10 pg/ml in all patients with cryptococcosis. The specificity of enolase antigen in the serodiagnosis of candidemia was 100%, but the sensitivity was 71.8%. The specificity and sensitivity of Cand-Tec, the assay for mannan antigen, and the assay for beta-glucan were 76.9 and 87.5%, 25.6 and 100%, and 84.4 and 87.5%, respectively. Our results demonstrated that antigen detection tests are useful for the diagnosis of candidemia; however, none is satisfactory for the serodiagnosis of candidemia. We suggest that a combination of two assays may increase the accuracy of diagnosis of candidiasis.