bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA fingerprinting and phenotyping of Mycobacterium tuberculosis isolates from human immunodeficiency virus (HIV)-seropositive and HIV-seronegative patients in Tanzania
Authors: Yang Z H , Mtoni I , Chonde M , Mwasekaga M , Fuursted K , Askgård D S , Bennedsen J , de Haas P E , van Soolingen D , van Embden J D
Published: January 2020

With the purpose of determining whether the risk of infection with a particular clone of Mycobacterium tuberculosis is influenced by the human immunodeficiency virus (HIV) status of the host, we analyzed and compared 68 mycobacterial isolates obtained from HIV-seropositive patients with tuberculosis (TB) in Dar es Salaam, Tanzania, with 66 mycobacterial isolates obtained from HIV-seronegative patients with TB in the same geographical region by using both DNA fingerprinting and classical phenotyping methods. One hundred one different IS6110 fingerprinting patterns were observed in the 134 isolates. The level of diversity of the DNA fingerprints observed in the HIV-seropositive group was comparable to the level of the diversity observed in the HIV-seronegative group. Resistance to a single anti-TB drug was found in 8.8% of the tested isolates, and 3.2% of the isolates were resistant to more than one anti-TB drug. The drug susceptibility profiles were not significantly difference between the two groups of isolates compared in the present study. Phenotypic characteristics which classify M. tuberculosis strains as belonging to the Asian subgroup correlated with a low IS6110 copy number per isolate. However, the occurrence of Asian subgroup strains was not associated with the HIV status of the patients. The results of the study suggested an equal risk of infection with a defined M. tuberculosis clone for HIV-seropositive and HIV-seronegative individuals.
Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870
Authors: Pfaller M A , Messer S A , Coffmann S
Published: January 2020

A study to compare three different methods for reading MIC endpoints tested by the broth microdilution modification of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) reference method was conducted. MICs of amphotericin B, flucytosine, fluconazole, itraconazole, and a new triazole, D0870, were determined for five reference yeast strains and 100 clinical isolates of Candida spp. MICs were read visually according to National Committee for Clinical Laboratory Standards guidelines from microdilution trays that had been (VS) and had not been (V) shaken. MICs were also determined spectrophotometrically (SP) at 492 nm. SP endpoints were determined as the concentrations resulting in a > or = 50% inhibition of growth (flucytosine and azoles) and a > or = 90% inhibition of growth (amphotericin B) relative to control growth. The five reference strains were tested nine times each against all five antifungal agents, and the MIC results for each reading method were compared with a 3-log2 dilution reference range determined by the macrodilution (M27-P) method. Overall, 84 to 100% of the MICs determined by V, 93 to 100% of those determined by VS, and 89 to 100% of those determined by SP fell within the 3-log2 dilution reference range for each reference strain and antifungal agent. Reproducibility was 99% for V and SP and 98% for VS. Agreement among the three methods of reading ranged from 97 to 99%. Excellent agreement among reading methods was also observed for all antifungal agents when tested against 100 clinical isolates. Agreement between the standard V method (no agitation) and VS ranged from 99 to 100%, and that between V and SP ranged from 89 to 99%. The VS and SP reading methods provided more definitive endpoints than the V method, which does not involve shaking.
Cluster of oral atypical Candida albicans isolates in a group of human immunodeficiency virus-positive drug users
Authors: Boerlin P , Boerlin-Petzold F , Durussel C , Addo M , Pagani J L , Chave J P , Bille J
Published: January 2020

Twenty-one chlamydospore-forming and germ tube-positive Candida albicans clinical isolates from 15 human immunodeficiency virus (HIV)-positive and 3 HIV-negative patients were examined by two different genetic methods. Multilocus enzyme electrophoresis and hybridization with the C. albicans-specific Ca3 probe showed that such isolates can be split into two genetically distinct groups that can be clearly distinguished. One group mainly contained strains with atypical sugar assimilation patterns and could be distinguished from the other group by the absence of intracellular beta-glucosidase activity. All 13 strains belonging to this group were isolated from the oral cavities of asymptomatic HIV-positive drug users and may be less pathogenic than the eight strains from the other group isolated either from HIV-positive patients with oropharyngeal candidiasis or from HIV-negative patients with invasive candidiasis.
Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR
Authors: Bretagne S , Costa J M , Marmorat-Khuong A , Poron F , Cordonnier C , Vidaud M , Fleury-Feith J
Published: January 2020

A competitive PCR assay involving the use of bronchoalveolar lavage (BAL) samples for the diagnosis of invasive pulmonary aspergillosis (IPA) was developed. For this purpose, a 1-kb mitochondrial DNA fragment of Aspergillus fumigatus was sequenced. The primers used allowed amplification of A. fumigatus, A. flavus, A. terreus, and A. niger DNAs but not DNAs of other fungi and yeasts. BAL samples from 55 consecutively enrolled patients were tested. Three samples were excluded because of failure of correct amplification of the internal competitive control. Of 28 immunocompromised patients, 6 were PCR positive; 3 died of IPA and their BAL cultures yielded A. fumigatus; and 3 were culture negative and did not develop IPA. Of 15 human immunodeficiency virus-positive patients and 9 immunocompetent patients, 5 and 4, respectively, were both PCR positive and culture negative, and none developed aspergillosis. Thus, PCR confirmed IPA in three patients but gave positive results for 25% (12 of 49) of the patients who did not develop aspergillosis. The predictive value of PCR-positive results seems low for patients at risk for aspergillosis. Moreover, the risk of contamination of reaction buffers or biological samples with Aspergillus conidia seems high and has to be weighed in regard to the potential diagnostic benefit of PCR testing as a routine procedure.
PCR amplicon restriction endonuclease analysis of the chromosomal dhps gene of Neisseria meningitidis: a method for studying spread of the disease-causing strain in contacts of patients with meningococcal disease
Authors: Kristiansen B E , Fermér C , Jenkins A , Ask E , Swedberg G , Sköld O
Published: January 2020

We tested two sets of primers derived from the dhps gene of Neisseria meningitidis for the amplification of meningococcal DNA by PCR. Both the NM1-NM6 primers and the NM3-NM6 primers amplified dhps DNA from all of the meningococci included in the study, resulting, in most cases, in amplicons of 0.70 and 0.23 kb, respectively. Also, dhps DNAs of N. gonorrhoeae and some commensals were amplified but Haemophilus influenzae, Streptococcus pneumoniae, and Escherichia coli DNAs were not. By PCR amplicon restriction endonuclease analysis (AREA) of the larger amplicon, we could differentiate between individual strains of N. meningitidis. Following two cases of meningococcal disease, we used PCR AREA to identify healthy contacts carrying the disease-causing strain. We conclude that PCR AREA is a useful method for meningococcal strain differentiation and that it has potential as a method for studying the spread of a disease-causing strain in an affected population. The method is quicker and easier to perform and interpret than chromosomal DNA fingerprinting.
Influence of hepatitis C virus (HCV) genotypes on HCV recombinant immunoblot assay patterns
Authors: Pawlotsky J M , Roudot-Thoraval F , Pellet C , Aumont P , Darthuy F , Remire J , Duval J , Dhumeaux D
Published: January 2020

Ninety-six patients with chronic hepatitis C were studied. A second-generation recombinant immunoblot assay detected anti-NS4 antibodies significantly more often in patients infected by hepatitis C virus genotype 1 than in patients infected by other types. By a third-generation recombinant immunoblot assay, the prevalences of the four antibodies measured did not differ according to the hepatitis C virus genotype.
Differentiation of pathogenic Escherichia coli strains in Brazilian children by PCR
Authors: Tornieporth N G , John J , Salgado K , de Jesus P , Latham E , Melo M C , Gunzburg S T , Riley L W
Published: January 2020

A PCR technique to differentiate pathogenic enteric Escherichia coli strains in a field setting was evaluated. Among 76 children with acute diarrhea, this technique identified 12 children (16%) with enterotoxigenic E. coli, 6 (8%) with enteropathogenic E. coli, and 1 (1%) with enteroinvasive E. coli infection. Compared with the conventional assays, the PCR method proved to be simpler, more rapid, and inexpensive and therefore suitable for application in a developing-country field setting.
Comparison of a direct antigen enzyme immunoassay, Herpchek, with cell culture for detection of herpes simplex virus from clinical specimens
Authors: Verano L , Michalski F J
Published: January 2020

Direct enzyme immunoassay (Herpchek) was compared with culture on 21,522 specimens mainly from asymptomatic women for herpes simplex virus detection. Sensitivity and specificity were 73.8 and 97.7%, respectively. The 33% detection rate by enzyme immunoassay in 5 h increased to 43% when the enzyme immunoassay was combined with culture. Herpchek alone was not sensitive enough, but in combination with culture, maximum detection was achieved.
Detection of antibodies against human papillomavirus (HPV) type 16 virions by enzyme-linked immunosorbent assay using recombinant HPV 16 L1 capsids produced by recombinant baculovirus
Authors: Le Cann P , Touze A , Enogat N , Leboulleux D , Mougin C , Legrand M C , Calvet C , Afoutou J M , Coursaget P
Published: January 2020

The L1 major capsid protein of human papillomavirus type 16 (HPV-16) was expressed in Sf-21 insect cells with a recombinant baculovirus. Virus-like particles obtained were purified and used to develop an enzyme-linked immunosorbent assay for detection of anti-HPV-16 antibodies in sera from 76 women with evidence of genital HPV infection and 79 controls. HPV-16-infected individuals developed antibodies directed at HPV-16 virions since reactivity against recombinant HPV-16L1 capsids was observed in 50% of them compared with only 6% in the general adult population. However, some cross-reactivities with sera from women infected with others HPV types were observed.
High specificity of Trypanosoma cruzi epimastigote ribonucleoprotein as antigen in serodiagnosis of Chagas' disease
Authors: Solana M E , Katzin A M , Umezawa E S , Miatello C S
Published: January 2020

We assessed the performance of an enzyme-linked immunosorbent assay (ELISA) with the Trypanosoma cruzi epimastigote ribosomal fraction (Tulahuen and Y strains) in order to improve the diagnostic specificity of the test. A total of 100 serum samples from patients with chronic Chagas' disease from Brazil and Argentina were studied. Sera from 116 patients, without Chagas' disease, including 10 with active mucocutaneous leishmaniasis and 20 with visceral leishmaniasis, were used as controls. Immunoglobulin G (IgG) antibodies against the ribosomal fraction (ribonucleoproteins [RNPs]) in the ELISA were found in 97% of samples from patients with Chagas' disease. A total of 99% of the sera from patients without the disease were negative, including sera from patients with mucocutaneous and visceral leishmaniases. The distribution of IgG isotypes in randomly chosen serum samples was determined by ELISA; IgG1 and IgG3 were predominant (100% exhibited IgG1 and 85% exhibited IgG3, and 50% also presented the IgG2 isotype. The distribution of the IgG subclasses was confirmed by the Western blot (immunoblot) technique. When total IgG was assayed by Western blot assay, no correlation was found between the pattern of serum reactivity and the clinical features of the patients with Chagas' disease. Therefore, no typical profile of polypeptide recognition could be associated with any clinical form of Chagas' disease (cardiomyopathy or megaviscera). Our results showed that sera from patients with Chagas' disease react with ribosomal antigens and display a typical profile of IgG isotypes (IgG1 plus IgG3). The RNP ELISA seems to have improved specificity compared with those of routine techniques such as the indirect immunofluorescence assay and hemagglutination because it better discriminates between patients with Chagas' disease and patients without the disease. Since sera from patients with leishmaniasis failed to show cross-reactivity with this antigen, the ELISA seems useful for detecting Chagas' disease as well as confirming the nature of sera, when it is doubtful whether the patients has Chagas' disease, by the isotype distribution of IgG.
Direct determination of cryptococcal antigen in transthoracic needle aspirate for diagnosis of pulmonary cryptococcosis
Authors: Liaw Y S , Yang P C , Yu C J , Chang D B , Wang H J , Lee L N , Kuo S H , Luh K T
Published: January 2020

Pulmonary cryptococcosis causes significant morbidity and mortality in immunocompromised patients. Definitive diagnosis of pulmonary cryptococcosis is usually difficult. The use of direct determination of cryptococcal antigen in transthoracic needle aspirate to diagnose pulmonary cryptococcosis was investigated. Over a 2-year period, we studied a total of 41 patients with respiratory symptoms and pulmonary infiltrates of unknown etiology who were suspected of having pulmonary cryptococcosis. Twenty-two patients were immunocompetent patients and 19 patients were immunocompromised. A diagnosis of pulmonary cryptococcosis was based on cytological examination, culture for Cryptococcus neoformans, histopathologic examination, and clinical response to antifungal therapy. All patients underwent chest ultrasound and ultrasound-guided percutaneous transthoracic needle aspiration to obtain specimens for cryptococcal antigen determination. The presence of cryptococcal antigen was determined by the latex agglutination system (CALAS; Meridian Diagnostics, Cincinnati, Ohio). An antigen titer equal to or greater than 1:8 was considered positive. The specimens were also sent for cytological examination, fungal culture, and/or histopathologic examination. A final diagnosis of pulmonary cryptococcosis was made in eight patients. Direct determinations of cryptococcal antigen in lung aspirate were positive in all eight patients with pulmonary cryptococcosis (100% sensitivity, 97% specificity, a positive predictive value of 89%, and negative value of 100%), and there was only one false-positive in noncryptococcosis patients. The diagnostic accuracy was 97.5%. Serum cryptococcal antigen was positive in only three patients with pulmonary cryptococcosis (sensitivity, 37.5%). This study showed that direct measurement of cryptococcal antigen in lung aspirate can be a rapid and useful test for diagnosis of pulmonary cryptococcosis.
Genotyping of hepatitis C virus in South Africa
Authors: Smuts H E , Kannemeyer J
Published: January 2020

The six major hepatitis C virus genotypes were investigated by using samples from 79 seropositive and PCR-positive blood donors from three different regions of South Africa as well as 9 patients with chronic renal failure, 19 with liver disease, and 23 with hemophilia. PCR products of the genome were typed by restriction fragment length polymorphic analysis by RsaI-HaeIII and MvaI-HinfI double digestion. Type 5 occurred in 40% of this population group; type 1 occurred in 33%; and types 2, 3, and 4 were found in 13.8, 7.7, and 2.3%, respectively.
Subdivision of Burkholderia pseudomallei ribotypes into multiple types by random amplified polymorphic DNA analysis provides new insights into epidemiology
Authors: Haase A , Smith-Vaughan H , Melder A , Wood Y , Janmaat A , Gilfedder J , Kemp D , Currie B
Published: January 2020

Ribotyping has previously been used for epidemiological studies of Burkholderia pseudomallei (previously Pseudomonas pseudomallei). We show here that random amplified polymorphic DNA (RAPD) analysis allows subdivision of strains of the same ribotype. With five different primers, no two epidemiologically unrelated isolates of any single ribotype in this study of 102 isolates from humans, goats, cats, and soil had identical RAPD patterns. Conversely, RAPD analysis showed clonality for isolates from each of two animal outbreaks of melioidosis and from a nontropical focus of animal and human melioidosis spanning 25 years. Some soil isolates were identical to epidemiologically related animal and human isolates as determined by RAPD typing. There was no evidence that the clinical outcome of melioidosis was related to RAPD patterns.
Rapid diagnosis of pulmonary tuberculosis by using Roche AMPLICOR Mycobacterium tuberculosis PCR test
Authors: D'Amato R F , Wallman A A , Hochstein L H , Colaninno P M , Scardamaglia M , Ardila E , Ghouri M , Kim K , Patel R C , Miller A
Published: January 2020

A rapid PCR-based test for the diagnosis of pulmonary tuberculosis, the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB), was evaluated. Results from AMPLICOR MTB were compared with culture results and the final clinical diagnosis for each patient. A total of 985 specimens from 372 patients were tested. When AMPLICOR MTB results were compared with resolved results, i.e., a specimen grew M. tuberculosis or was obtained from a patient with a clinical diagnosis of tuberculosis, the sensitivity, specificity, positive predictive value, and negative predictive value for the AMPLICOR MTB test were 66.7, 99.6, 91.7, and 97.7%, respectively. These results were comparable to those obtained from culture. Test results were available approximately 6.5 h after specimen receipt in the laboratory. Our data demonstrate that AMPLICOR MTB will provide rapid, valuable information for the diagnosis and control of tuberculosis.
Isolation and characterization of a naturally occurring parainfluenza 3 virus variant
Authors: Swierkosz E M , Erdman D D , Bonnot T , Schneiderheinze C , Waner J L
Published: January 2020

A parainfluenza 3 virus variant which failed to react with parainfluenza 3 virus-specific monoclonal anti-bodies from two commercial sources was isolated from a 14-month-old boy. Analysis of the coding region of the hemagglutinin-neuraminidase gene identified 36 nucleotide changes and 4 amino acid changes compared with a consensus sequence derived from strains isolated from 1957 through 1983. Two unique amino acid changes occurred at positions 174 and 283, which are close to identified epitopes in the hemagglutinin-neuraminidase protein. Ongoing viral surveillance to detect variants is important, particularly in regard to vaccine development.
Genetic variability of Bacillus anthracis and related species
Authors: Harrell L J , Andersen G L , Wilson K H
Published: January 2020

We evaluated the abilities of pulsed-field gel electrophoresis (PFGE) and sequences of intergenic spacer regions (ISRs) between two highly conserved genes, 16S-23S rDNA and gyrB-gyrA ISRs, to detect variation in strains of Bacillus anthracis as well as two closely related species, B. cereus ATCC 14579 and B. mycoides ATCC 6462. For each restriction enzyme, (NotI, SfiI, and SmaI), the PFGE banding patterns for three B. anthracis strains (Ames, Vollum, and Sterne) were identical. However, closely related species could be differentiated from B. anthracis and from each other. PCR amplification of the 16S-23S rDNA ISR yielded a 143- to 144-bp fragment, showing identical sequences for B. anthracis strains, one nucleotide deletion between B. cerus and B. anthracis, and 13 nucleotide differences between B. mycoides and B. anthracis. The gyrase ISR sequences (121 bp) in B. anthracis strains were also identical, but those in B. cereus and B. mycoides differed from that in B. anthracis by 1 and 2 nucleotides, respectively, and from each other by only 1 nucleotide. Given the diverse geographic origins of these B. anthracis strains, this species is very homogenous. We conclude that methods such as PFGE and sequences of ISRs may be useful in separating B. anthracis from closely related species, but more sensitive methods are needed for strain identification of B. anthracis.
Identification of Bartonella (Rochalimaea) species among fastidious gram-negative bacteria on the basis of the partial sequence of the citrate-synthase gene
Authors: Joblet C , Roux V , Drancourt M , Gouvernet J , Raoult D
Published: January 2020

The bacterial genus Bartonella (Rochalimaea) includes emerging human pathogens with five recognized species. These are fastidious gram-negative bacteria, exhibiting few phenotypic characteristics and whose identification relies upon serotyping, cellular fatty acid analysis, and molecular typing. Most of the isolates have been recovered from the blood of patients, and three of the four pathogenic Bartonella species are associated with infectious endocarditis. We performed PCR-restriction fragment length polymorphism (RFLP) analysis of the blood culture bottle supernatant for the routine identification of Bartonella species among fastidious gram-negative bacteria. The amplification of the citrate-synthase gene with primers previously reported (R. L. Regnery, C. L. Spruill, and B. D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) yielded a 379-bp product from Bartonella species and a 382-bp product for Capnocytophaga ochracea but no product from any of the other 15 genotypically or phenotypically related species tested. We determined the sequences of the citrate-synthase gene-amplified products for Bartonella species and C. ochracea in order to predict the optimal restriction enzyme to be used in RFLP analysis. TaqI and AciI allowed identification of Bartonella species and C. ochracea. We propose that acridine orange and Gram staining, followed by PCR-RFLP analysis of the blood bottle supernatant, be included in the examination of blood samples from patients with suspected infectious endocarditis.
Evidence that the National Committee for Clinical Laboratory Standards disk test is less sensitive than the screen plate for detection of low-expression-class methicillin-resistant Staphylococcus aureus
Authors: Mackenzie A M , Richardson H , Lannigan R , Wood D
Published: January 2020

A low-expression-class methicillin-resistant Staphylococcus aureus strain (strain 9302-2) was sent to 207 laboratories as part of the bacteriology component of the Laboratory Proficiency Testing Program of Ontario. An incorrect (susceptible) result was reported by 16 of 76 (21%) of laboratories that used the National Committee for Clinical Laboratory standards disk test, whereas 1 of 104 laboratories that used other methods reported an incorrect result (P < 0.05). Experiments showed discrepancies in the disk test results given by Mueller-Hinton agars from three manufacturers. We advise that laboratories should use a low expression-class methicillin-resistant S. aureus isolate as a control for the National Committee for Clinical Laboratory Standards disk test.
Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts
Authors: Petry F , Robinson H A , McDonald V
Published: January 2020

Propagation of Cryptosporidium parvum is problematic because in vitro development of the parasite is poor and animals are only briefly susceptible as neonates. At present oocysts of the parasite are usually procured by passage in neonatal sheep or cattle. In the present study, large numbers of oocysts of C. parvum could be isolated following infection of dexamethasone-treated adult C57BL/6 mice. The amount of immunosuppressive drug and the regimen of administration were critical for successful maintenance of the parasite, however. Routinely, 10 mice (age, 8 to 12 weeks) were injected four times on alternate days with 1.0 mg of dexamethasone, and the last injection was given on the same day as oral inoculation with 10(6) oocysts. By using a simplified procedure for oocyst purification from mouse feces, approximately 10(9) oocysts were obtained. This model is inexpensive and comparatively safe to handle, and the numbers of animals inoculated can be varied to obtain the required number of oocysts. Thus, this murine infection model would be a suitable alternative to the use of neonatal calves or sheep for efficient oocyst propagation.
Isolation of Porphyromonas gingivalis strain from tubal-ovarian abscess
Authors: Hirata R , Ménard C , Fournier D , Catellani M A , Mouton C , Ferreira M C
Published: January 2020

An unusual case of involvement of Porphyromonas gingivalis is described. Two anaerobic isolates, identified as Fusobacterium nucleatum and P. gingivalis, were recovered from the pus of a tubal-ovarian abscess in a 35-year-old woman. Identification of the P. gingivalis isolate was confirmed by randomly amplified polymorphic DNA fingerprinting.
Analysis of Salmonella typhi isolates from Southeast Asia by pulsed-field gel electrophoresis
Authors: Thong K L , Puthucheary S , Yassin R M , Sudarmono P , Padmidewi M , Soewandojo E , Handojo I , Sarasombath S , Pang T
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) revealed that multiple genetic variants of Salmonella typhi are simultaneously present in Southeast Asia and are associated with sporadic cases of typhoid fever and occasional outbreaks. Comparative analysis of PFGE patterns also suggested that considerable genetic diversity exists among S. typhi strains and that some PFGE patterns are shared between isolates obtained from Malaysia, Indonesia, and Thailand, implying movement of these strains within these regions of Southeast Asia, where they are endemic.
Evaluation of new rapid commercial enzyme immunoassay for detection of Cryptosporidium oocysts in untreated stool specimens
Authors: Parisi M T , Tierno P M
Published: January 2020

Sixty-six stool specimens were evaluated by the ProSpecTR Cryptosporidium rapid enzyme immunoassay (EIA) (Alexon, Sunnyvale, Calif.). Approximately 2 g of untreated stool suspended in buffer was filtered through membranes labelled with anti-Cryptosporidium-specific antigen antibody. Anti-Cryptosporidium-specific antigen antibody was labelled with biotin, horseradish peroxidase conjugated to streptavidin, and tetramethylbenzidine, and each labelled antibody was added in sequence to the membranes. Each membrane had a positive control and test area. EIA results were compared with those of the modified acid-fast procedure. Twenty-three specimens were positive by the initial acid-fast procedure and the EIA. Forty-two specimens were negative by the initial acid-fast test and the EIA. One specimen was negative by the initial acid-fast test and positive by the EIA (sensitivity, 100%; specificity, 98.5%). This technique is easy to use by comparison with the cumbersome, labor-intensive, and more subjective microscopic methods currently available, and its sensitivity equals that of current microscopic methods.
Human herpesviruses 6 and 7 in cervixes of pregnant women
Authors: Okuno T , Oishi H , Hayashi K , Nonogaki M , Tanaka K , Yamanishi K
Published: January 2020

We looked for human herpesvirus 6 (HHV-6) and HHV-7 genomes in the cervixes of pregnant women in the late stages of their pregnancies. Of 72 samples collected with cervical swabs and amplified by nested PCR, we found that 14 (19.4%) and 2 (2.7%) contained detectable HHV-6 and HHV-7 genomes, respectively. The two samples in which HHV-7 DNA was detected also contained HHV-6 genomes. Hybridization of HHV-6 DNA amplified by PCR with variant-specific probes revealed that all of these DNA samples belonged to variant type B. These results indicated that HHV-6 and/or HHV-7 exists in the cervixes of infected women in late pregnancy and may cause perinatal infection.
Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay
Authors: Coutlée F , Provencher D , Voyer H
Published: January 2020

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.
Diagnostic difficulties caused by a nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung
Authors: Sigler L , de la Maza L M , Tan G , Egger K N , Sherburne R K
Published: January 2020

The presence of clamp connections on hyphae and the development of fruiting bodies in culture are primary characters which allow identification of the basidiomycete Schizophyllum commune in cases of human infection. The diagnostic problems presented by a nonclamped, nonfruiting isolate from a dense mass in the right upper lobe of the lung in a female with a past history of pulmonary tuberculosis and diabetes are described. Several features of the isolated fungus, including rapid growth rate and white, dense, cottony colonies, tolerance to the fungicide benomyl at a concentration of 10 micrograms/ml, and susceptibility to cycloheximide at 400 micrograms/ml, suggested that it might be a basidiomycete. Transmission electron microscopy showed the presence of a dolipore septum with perforate pore cap characteristic of fungi in the class Holobasidiomycetes. However, species identification remained elusive until compatibility tests with known single-basidiospore isolates confirmed the identification of the sterile lung isolate as S. commune. Sequence analysis of the 5' internal transcribed spacer region of ribosomal DNA further supported conspecificity.
Evaluation of commercial antisera for Shigella serogrouping
Authors: Lefebvre J , Gosselin F , Ismaïl J , Lorange M , Lior H , Woodward D
Published: January 2020

Shigella serogrouping antisera from six companies (Becton Dickinson, Denka, Difco, Murex, Roach, and Sanofi-Pasteur) intended for the slide agglutination test and those of the Wellcolex Colour Shigella latex agglutination test were evaluated to identify quality products for Shigella identification. Forty-six reference Shigella strains (one for each serotype and species), 50 clinical strains (21 S. flexneri, 21 S. sonnei, 4 S. dysenteriae, 4 S. boydii) representing the most prevalent species and serotypes encountered in Quebec, and 9 non-Shigella strains were tested according to the manufacturers' instructions. A 3+ reaction (> or = 75% agglutination) was considered positive for the slide agglutination tests. Sensitivity varied from 47% (Roach) to 94% (Difco). For the 105 strains tested, accuracy ranged from 53% (Roach) to 91% (Wellcolex). Specificity varied from 97 to 100% for group A antisera, from 96 to 100% for group B antisera, from 88 to 100% for group C antisera, and from 95 to 99% for group D antisera. The costs of reagents required to test one strain varied from $3.50 to $13.20 (in Canadian dollars). In conclusion, Roach reagents proved to be unsatisfactory for Shigella serogrouping. Among those from the remaining companies, the Denka, Difco, and Wellcolex reagents met a performance standard of 90% accuracy.
Effect of PANTA on growth of Mycobacterium kansasii in BACTEC 12B medium
Authors: Conville P S , Andrews J W , Witebsky F G
Published: January 2020

Mycobacterium kansasii isolates from two patients showed relatively slow growth in BACTEC 12B medium (12B) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) compared with the more rapid growth of these isolates on solid media. This finding prompted an evaluation of the effect of PANTA (Becton Dickinson) on the growth rate of these isolates. Suspensions of one isolate from each of these two patients (A and B), six additional isolates from six other patients (C through H), and one M. kansasii American Type Culture Collection isolate were inoculated into 12B with PANTA, 12B with reconstituting fluid only, and Middlebrook 7H11 agar plates (Remel, Lenexa, Kans.). For the isolates from patients A and B, the average times to detection for 12B with PANTA, 12B with reconstituting fluid, and Middlebrook 7H11 agar were 12.3, 7.4, and 9.0 days, respectively. For the remaining six patient isolates and the American Type Culture Collection strain, the average times to detection for these media were 9.2, 8.1, and 9.6 days. Susceptibility tests performed with the isolates from patients A and B with the individual component antibiotics of PANTA and testing of four of the other isolates with nalidixic acid alone suggested that nalidixic acid exerts some degree of inhibition on the growth of M. kansasii. The eight patient isolates were also inoculated onto Lowenstein Jensen medium (Remel) and onto a variety of selective mycobacterial media containing nalidixic acid and other antimicrobial agents. All isolates showed some degree of inhibition on at least one of these selective media.
PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital
Authors: Martirosian G , Kuipers S , Verbrugh H , van Belkum A , Meisel-Mikolajczyk F
Published: January 2020

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique. The other strain commonly encountered among the neonates was also identified frequently among the isolates from patients with antibiotic-associated diarrhea or pseudomembranous colitis, indicating its general occurrence. On the basis of both epidemiological studies and PCR-mediated genotyping, it was shown that the environment and not the birth canal is the major source of C. difficile acquisition by neonates in this maternity hospital setting. Furthermore, AP-PCR appears to be a fast and useful method for epidemiologically relevant typing of C. difficile isolates.
PCR strategy for identification and differentiation of small pox and other orthopoxviruses
Authors: Ropp S L , Jin Q , Knight J C , Massung R F , Esposito J J
Published: January 2020

Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.
Detection kinetics for positive blood culture bottles by using the VITAL automated system
Authors: Marchandin H , Compan B , Simeon De Buochberg M , Despaux E , Perez C
Published: January 2020

The VITAL system principle is based on homogeneous fluorescence technology. During an 11-month period, a total of 19,706 blood cultures from adult patients hospitalized in various establishments of the Montpellier Teaching Hospital were collected in VITAL bottles, of which 1,939 were declared positive. Only 204 bottles (1.04%) were false positives. The 1,735 true-positive bottles were collected from 130 patients. The final visual control permitted the detection of 10 falsely negative bottles (0.05%), of which 5 contained clinically significant microorganisms from four patients. The kinetics of detection for all microorganisms showed that 66.6% were detected within 24 h, 83.1% within 48 h, 95.5% within 120 h, and 100% within 150 h. No clinical episode would have been missed had a 5-day protocol been used instead of a 7-day protocol. Among the positive bottles, 65.7% were detected by the SLOPE algorithm, 20.1% by the DELTA algorithm, and 14.2% by the THRESHOLD algorithm. This retrospective study of our results shows that a 5-day protocol is sufficient for the detection of septic episodes using the VITAL system.
Molecular method for typing Mycobacterium ulcerans
Authors: Jackson K , Edwards R , Leslie D E , Hayman J
Published: January 2020

A cluster of Mycobacterium ulcerans infections has recently occurred on Phillip Island, Victoria, Australia. Previous cases of infection have generally been located around Bairnsdale in southeast Gippsland. The aim of this study was to determine the epidemiological relationship between these strains and other strains originating in Australia and Africa. The previously described plasmid pTBN12 was used as a probe with restriction enzyme-digested chromosomal DNA to differentiate the strains of M. ulcerans. The probe was able to distinguish 11 restriction fragment length polymorphisms (RFLPs). Forty-three strains originating in Victoria were divided into three types, i.e., V1, V2, and V3. The majority of strains (40) yielded a type V1 pattern, including strains from southeast Gippsland. Fourteen strains from Queensland yielded three additional RFLP types, i.e., Q1, Q2, and Q3. Five strains from Benin and seven strains from Zaire yielded five additional RFLP types. It is envisaged that molecular typing of M. ulcerans strains from around the world may have a great impact on understanding of the epidemiology of infection with this organism.
Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp.?
Authors: Kent L , McHugh T D , Billington O , Dale J W , Gillespie S H
Published: January 2020

The insertion sequence IS6110 has an important role in diagnostic PCR and typing of Mycobacterium tuberculosis. We have evaluated a one-tube nested PCR which detects IS6110. Positive results were obtained with DNAs from four of four M. tuberculosis isolates, seven of eight M. fortuitum isolates, seven of seven M. avium-M. intracellulare complex isolates, four of five M. kansasii isolates, four of five M. xenopi isolates, two of four M. malmoense isolates, and one of two M. chelonei isolates. These results were confirmed by hybridization of genomic DNA from bp 505 to 685 of the IS6110 from M. tuberculosis H37Rv. Dot blot hybridization of genomic DNAs from these isolates with the same probe cinfirmed the presence of a homologous sequence in these mycobacterial species. These data suggest that false-positive results may be obtained for clinical samples when some methods based on IS6110 are used [corrected].
Use of random amplified polymorphic DNA as a typing method for Candida albicans in epidemiological surveillance of a burn unit
Authors: Robert F , Lebreton F , Bougnoux M E , Paugam A , Wassermann D , Schlotterer M , Tourte-Schaefer C , Dupouy-Camet J
Published: January 2020

Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hôpital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. albicans strains isolated from various anatomical sites of unrelated patients, and the strains showed 22 different patterns. Random amplified polymorphic DNA was then used in the epidemiological surveillance of the patients in the burn unit over a 9-month period. Seven patterns were identified among 84 isolates from 18 patients. One pattern (pattern A) corresponding to isolates from 7 of the 18 patients (68% of isolates) predominated throughout the 9-month study, while some strains with other profiles were isolated only once. Some profiles appeared to show a particular geographic pattern within the unit, suggesting transmission from room to room. These results underline the importance of fungal surveillance in such patients and the need to inform nursing staff of measures to prevent the spread of Candida spp. from patient to patient.
Evaluation of Cobas Core Rubella IgG EIA recomb, a new enzyme immunoassay based on recombinant rubella-like particles
Authors: Grangeot-Keros L , Pustowoit B , Hobman T
Published: January 2020

Cobas Core Rubella IgG EIA recomb (Roche), a new, commercially available rubella enzyme immunoassay using recombinant rubella-like particles, was compared with a hemagglutination inhibition assay (Rubenosticon; Organon Teknika) and two whole-virus enzyme immunoassays (IMx [Abbott] and Platelia [Sanofi Diagnostics Pasteur]). Compared with those of these reference assays, the relative sensitivities of Cobas Core Rubella IgG recomb were 100, 94, and 95.9%, with specificities amounting to 80.8, 98, and 98.2%, respectively.
Characterization of chromosomal DNA profiles from Helicobacter pylori strains isolated from sequential gastric biopsy specimens
Authors: Salama S M , Jiang Q , Chang N , Sherbaniuk R W , Taylor D E
Published: January 2020

The restriction endonuclease profiles of DNAs from Helicobacter pylori strains isolated from 20 patients in two or more consecutive biopsy specimens over a period of up to 2 years were analyzed by pulsed-field gel electrophoresis with NotI and NruI. H. pylori strains possess a high degree of genomic diversity which was not observed to occur in vivo, and attempts to observe it in vitro were not successful.
Comparison of recovery rates for mycobacteria from BACTEC 12B vials, Middlebrook 7H11-selective 7H11 biplates, and Lowenstein Jensen slants in a public health mycobacteriology laboratory
Authors: Wilson M L , Stone B L , Hildred M V , Reves R R
Published: January 2020

Recovery rates from Middlebrook 7H11-selective 7H11 biplates and Lowenstein-Jensen slants (LJ) used with BACTEC 12B vials were compared for 5,399 specimens. For 578 specimens that were inoculated onto three media, 580 mycobacteria were isolated, including 277 (48%) Mycobacterium avium complex isolates, 230 (40%) Mycobacterium tuberculosis isolates, and 73 (12%) other mycobacteria. For BACTEC 12B vials, 506 (87%) cultures were positive, 45 (8%) were negative, and 29 (5%) were lost to contamination; for 7H11-7H11-selective biplates, 469 (81%) cultures were positive, 95 (16%) were negative, and 16 (3%) were lost to contamination; for LJ, 230 (40%) cultures were positive, 111 (19%) were negative, and 239 (41%) were lost to contamination. For routine cultures, use of plate media is superior to use of LJ.
Outbreak of group A streptococci in a burn center: use of pheno- and genotypic procedures for strain tracking
Authors: Gruteke P , van Belkum A , Schouls L M , Hendriks W D , Reubsaet F A , Dokter J , Boxma H , Verbrugh H A
Published: January 2020

In a burn center, an outbreak of group A streptococci (GAS) colonizations involving 13 patients and two staff members occurred. Adverse events due to GAS, loss of skin graft after initial take (secondary loss) and partial take, occurred in patients who underwent surgery before the colonization was detected. GAS isolates from nine patients and one staff member were stored and subsequently pheno- and genotyped by T serotyping, gas chromatography, M genotyping, and random amplified polymorphic DNA typing. The outbreak was caused by two types of GAS, identified as T4/28-M48 and T13-M77 by T serotyping and M genotyping. Random amplified polymorphic DNA typing and gas chromatography distinguished both clusters accurately. One subcluster indicated by gas chromatography could be linked to the acquisition of GAS from a roommate. The T13-M77 cluster was characterized by rapid spread through the center compared with the T4/28-M48 cluster. One patient contracted the T13-M77 strain while in protective isolation, indicating a role for staff members in the transmission. Our standard GAS control policy, consisting of twice weekly screening of all burned patients and immediate isolation and treatment, proved efficacious in preventing further spread of GAS. Reporting by staff members of signs and symptoms compatible with GAS infection was reenforced.