bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of molecular typing methods and antifungal susceptibility testing to the study of a candidemia cluster in a burn care unit
Authors: Bart-Delabesse E , van Deventer H , Goessens W , Poirot J L , Lioret N , van Belkum A , Dromer F
Published: January 2020

We investigated a cluster of cases of Candida septicemia diagnosed in four burn patients. Twenty clinical isolates of Candida albicans and two of Candida parapsilosis, plus eight isolates of C. albicans recovered from nurses' clothes, were analyzed by antifungal susceptibility testing and three genotyping methods (restriction fragment length polymorphism analysis with EcoRI and HinfI, arbitrarily primed PCR, and karyotyping). The high MICs of the azoles for all of the C. albicans isolates tested suggest either a natural resistance of the endogenous flora or the transmission of isolates with acquired resistance. The genotyping methods demonstrated the involvement of four different strains, cross-infections with one C. albicans strain and one C. parapsilosis strain, and identity between some of the strains from the patients and nurses. The origins of the strains remain unclear. Our results show that the use of a combination of at least two different methods such as those used in the present study is recommended for C. albicans typing.
PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection
Authors: Nelson C T , Istas A S , Wilkerson M K , Demmler G J
Published: January 2020

PCR detected cytomegalovirus (CMV) DNA in the serum of 18 of 18 infants with symptomatic congenital CMV infection, 1 of 2 infants with asymptomatic congenital CMV infection, and 0 of 32 controls. Serum CMV PCR provided a rapid, sensitive, and specific method for diagnosis of congenital CMV infection in infants who were symptomatic at birth.
Simple enzyme immunoassay for titration of antibodies to the CD4-binding site of human immunodeficiency virus type 1 gp120
Authors: Turbica I , Posner M , Bruck C , Barin F
Published: January 2020

We report the development of an immunoassay for the titration of antibody to the CD4-binding site (CD4BS) of the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120. This assay is a competitive enzyme-linked immunosorbent assay in which serum antibodies compete with labeled F105, a human monoclonal antibody whose corresponding epitope overlaps the conformation-dependent CD4BS, for binding to purified recombinant gp120 coated on a solid phase. Ninety-nine percent (109 of 110) of HIV-1-positive French patients and 91% (51 of 56) of HIV-1-positive African patients had CD4BS antibodies, indicating that the conformational CD4BS epitope is well conserved among different subtypes of HIV-1. Titers of CD4BS antibodies according to clinical status appeared to be not statistically different. A longitudinal study in 21 seroconverters showed that, for the majority of individuals, CD4BS antibodies appeared early and persisted at relatively high titers for several years. None of 21 HIV-2-seropositive patients had CD4BS antibodies in our assay, suggesting that the antibodies produced during HIV-2 infection are not cross-reactive with the CD4BS of HIV-1 gp120.
Mucormycosis caused by Rhizopus microsporus var. microsporus: cellulitis in the leg of a diabetic patient cured by amputation
Authors: West B C , Oberle A D , Kwon-Chung K J
Published: January 2020

Mucormycosis accompanied the development of bacterial infection in the leg of a diabetic African-American man. Local injury, diabetic ketoacidosis, renal insufficiency, and antimicrobial therapy were factors that contributed to the pathogenesis of the mucormycosis. The cellulitis was caused in part by Rhizopus microsporus var. microsporus and was cured by amputation. We report this unusual case of mucormycosis to emphasize the value of fungal identification, to illustrate a dramatic and successful clinical result, and to draw attention to an apparent role for bacterial infection and its treatment in the pathogenesis of mucormycosis. It is the third case report of mucormycosis in a human in which R. microsporus var. microsporus was definitively identified as the etiologic agent.
Quantification of hepatitis C virus RNA by competitive amplification of RNA from denatured serum and hybridization on microtiter plates
Authors: Ravaggi A , Zonaro A , Mazza C , Albertini A , Cariani E
Published: January 2020

The direct detection of hepatitis C virus (HCV) RNA by PCR is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. We report a technique of competitive amplification allowing the estimation of HCV RNA copy number in biological samples. We constructed a standard competitive RNA template containing only two point mutations compared with its wild-type counterpart. The competitor was added in titrated amounts to the target RNA, and the mixture was then reverse transcribed and amplified in the same reaction tube. The relative amounts of target and competitor were determined by differential hybridization on microtiter plates with nonradioactive probes. The evaluation of HCV RNA titer required a single coamplification with the competitor and could be read from a standard curve. Furthermore, this method proved suitable for amplification of HCV RNA directly from serum, thus avoiding the intrinsic variability of the RNA extraction step.
Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies
Authors: Starkey W G , Newcombe J , Corbett K M , Liu K M , Sanders P G , Best J M
Published: January 2020

Two glutathione S-transferase fusion proteins containing 44 (p1503) and 75 (p1509) amino acid residues of the rubella virus E1 glycoprotein were expressed in Escherichia coli with the aim of producing a recombinant rubella virus antigen for use in serological assays. p1503 contained three neutralizing and hemagglutinating epitopes (G. M. Terry, L. M. Ho-Terry, P. Londesborough, and K. R. Rees, Arch. Virol. 98:189-197, 1988); p1509 contained the putative neutralization domain described by Mitchell et al. (L. A. Mitchell, T. Zhang, M. Ho, D. Decarie, A. Tingle, M. Zrein, and M. Lacroix, J. Clin. Microbiol. 30:1841-1847, 1992) in addition to the three epitopes present in p1503. Both fusion proteins were soluble and affinity purified on glutathione-Sepharose 4B. In Western blots (immunoblots), p1503 and p1509 reacted with human sera containing rubella virus-specific immunoglobulin G. When used as antigens in indirect enzyme immunoassays to detect rubella virus-specific immunoglobulin G, p1503 correctly identified the rubella virus antibody status of 43 (76.8%) and p1509 correctly identified that of 48 (85.7%) of 56 serum samples received for routine rubella virus antibody screening. The results obtained with p1509 compare well with those obtained with a latex agglutination assay.
Report of six cases of human infection by Serratia plymuthica
Authors: Carrero P , Garrote J A , Pacheco S , García A I , Gil R , Carbajosa S G
Published: January 2020

Serratia plymuthica is an uncommon cause of human infection. Only one case of chronic osteomyelitis and two cases of sepsis secondary to central venous catheter infection have been documented. We report the isolation of S. plymuthica from six patients. The organism was recovered from blood cultures in three cases in which the patients had lymphoblastic leukemia, lymphoma, or stroke. Two isolates were recovered from exudates (following knee and abdominal surgery). In the last case, the organism was isolated from the peritoneal fluid of a patient with cholecystitis. The infection was considered nosocomial in five cases and community acquired in the other.
Measurement of human immunodeficiency virus type 1 p24 in serum by an ultrasensitive enzyme immunoassay, the two-site immune complex transfer enzyme immunoassay
Authors: Hashida S , Hashinaka K , Nishikata I , Oka S , Shimada K , Saitoh A , Takamizawa A , Shinagawa H , Ishikawa E
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) p24 antigen was measured by an ultrasensitive enzyme immunoassay (two-site immune complex transfer enzyme immunoassay). The antigen was reacted simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-anti-recombinant p24 (rp24) Fab' conjugate and anti-rp24 Fab'-beta-D-galactosidase conjugate. The complex that was formed, comprising the three components, was transferred from polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) immunoglobulin G (IgG) to polystyrene beads coated with streptavidin. The detection limit of rp24 was 2.4 fg (0.1 amol) per assay or 0.24 pg/ml with as little as 10 microliters of serum. When sera were treated at low pH, p24 was detected in 34 (68%) of 50 serum samples from asymptomatic carriers, in 25 (86%) of 29 serum samples from patients with advanced HIV-1 infection, and in none of 117 serum samples from HIV-1-seronegative individuals. Levels of p24 in serum were inversely correlated to those of anti-HIV-1 p24 IgG, and the recovery of rp24 added to serum decreased to zero with increasing levels of anti-HIV-1 p24 IgG in serum. This sensitive method may be used as a powerful tool for investigating the disease.
Excretion of bovine herpesvirus 1 in semen is detected much longer by PCR than by virus isolation
Authors: Van Engelenburg F A , Van Schie F W , Rijsewijk F A , Van Oirschot J T
Published: January 2020

To compare the sensitivities of PCR and virus isolation and to examine the course of virus excretion in semen, we intrapreputially inoculated eight bulls with bovine herpesvirus 1 (BHV1) and used two bulls as sentinels. From these bulls, we collected a large panel of semen samples during 65 days postinfection (dpi). At 44 dpi the bulls received dexamethasone to reactivate putatively latent virus. We analyzed the semen samples by virus isolation on egg yolk-extended semen (VIE test), by virus isolation on fresh semen (VIF test), and by a PCR test on egg yolk-extended semen. Of the 162 semen samples that were collected, the VIE test scored 24 positive, the VIF test scored 51 positive, and the PCR test scored 118 positive. At 6 dpi all samples from the inoculated bulls were found to be positive by all three tests. From 9 to 44 dpi most samples were found to be negative by both virus isolation tests but positive by the PCR test. From 48 to 55 dpi the dexamethasone treatment induced virus reactivation, which was evidenced by an increase in the number of positive VIE, VIF, or PCR tests. From 58 to 65 dpi all samples were found to be negative in both virus isolation tests, but several samples were still found to be positive by the PCR test. To determine whether BHV1 DNA was present in the dorsal root ganglia of the infected bulls, we analyzed by PCR several thoracic, lumbar, and sacral ganglia collected at 65 dpi. BHV1 DNA was frequently present in the third, fourth, and fifth sacral ganglia, and semiquantitative PCR analysis showed that the highest amounts of BHV1 DNA (10 to 30 molecules of BHV1 DNA per 10(5) cells) were present in the third sacral ganglion, The results demonstrate that the PCR test detected five times as many positive semen samples as the VIE test. Hence, intrapreputially infected bulls excrete BHV1 in semen much longer than recognized until now.
Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay
Authors: Ziegler T , Hall H , Sánchez-Fauquier A , Gamble W C , Cox N J
Published: January 2020

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.
Reverse transcriptase sequence of paired isolates of cerebrospinal fluid and blood from patients infected with human immunodeficiency virus type 1 during zidovudine treatment
Authors: Di Stefano M , Sabri F , Leitner T , Svennerholm B , Hagberg L , Norkrans G , Chiodi F
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) isolates obtained from the blood of patients undergoing treatment with 3'-azido-3'-deoxythymidine (zidovudine [AZT]) show a decreased sensitivity to the drug in vitro. The aim of the present study was to determine if HIV-1 variants resistant to AZT are present also in the brain compartment. We selected sequential HIV-1 isolates from the blood and the cerebrospinal fluid (CSF) of six patients with HIV-1 infection undergoing AZT therapy for a time varying between 1 and 3 years. The isolates were used to infect peripheral blood mononuclear cell cultures which were used to prepare viral DNA. The viral DNA was amplified by PCR and then directly sequenced. Analysis of the reverse transcriptase (RT) sequence of the isolates from the CSF during therapy demonstrated that CSF-resistant isolates are characterized by the same mutations documented in resistant isolates from the blood compartment. Isolates obtained from one patient (patient 3) showed the same two mutations (codons 70 and 215) in blood and CSF, whereas isolates obtained from an additional four patients presented a different pattern of mutations in the two compartments. We also analyzed the degree of amino acid homology between RT sequences from blood and CSF isolates in patients before and during AZT treatment. The percentages of amino acid variations were approximately equal when isolates from the same or different compartments were considered. Excluding the codons involved in AZT resistance, the time point of sampling did not affect RT variations during therapy significantly. In conclusion, our studies show that AZT-resistant HIV-1 can be found in the CSF of patients undergoing treatment. The mutations linked to AZT resistance in the CSF isolates are the same as those identified in AZT-resistant isolates from blood.
Restriction fragment length polymorphism of rRNA genes for molecular typing of members of the family Legionellaceae
Authors: Bangsborg J M , Gerner-Smidt P , Colding H , Fiehn N E , Bruun B , Høiby N
Published: January 2020

Typing of Legionella pneumophila remains important in the investigation of outbreaks of Legionnaires' disease and in the control of organisms contaminating hospital water. We found that the discriminatory power of a nonradioactive ribotyping method could be improved by combining results obtained with four restriction enzymes (HindIII, NciI, ClaI, and PstI). Fifty-eight clinical and environmental L. pneumophila strains including geographically unrelated as well as epidemiologically connected isolates were investigated. Epidemiologically related strains had the same ribotypes independent of the combinations of enzymes used. Some strains belonging to the same serogroup were assigned to different ribotypes, and some ribotypes contained members of different serogroups, indicating, as others have found, that serogroup and genotype are not always related. The discriminatory power of the method was estimated by calculating an index of discrimination (ID) for individual enzymes and combinations thereof. The combined result with all four enzymes was highly discriminatory (ID = 0.97), but results for three enzymes also yielded ID values acceptable for epidemiological purposes. In addition, the testing of 27 type strains and 6 clinical isolates representing Legionella species other than L. pneumophila indicated that ribotyping might be of value for species identification within this genus, as previously suggested.
Isolation of Coxiella burnetii from heart valves of patients treated for Q fever endocarditis
Authors: Mühlemann K , Matter L , Meyer B , Schopfer K
Published: January 2020

Coxiella burnetii was isolated from the valve material of two patients who underwent valvectomy because of progressive congestive heart failure due to endocarditis. In each case antibiotic therapy was administered for several months prior to valvectomy. Classical histopathological examination of the valves did not reveal an etiology. However, coxiella-like organisms were demonstrated in valvular material with Köster, Stamp, and Giemsa stains, and the organisms were grown in cell culture. Antibody titers were consistent with the diagnosis of chronic C. burnetii infection. This report illustrates the advantage of simple and fast staining techniques and cell culture for the demonstration and isolation of C. burnetii in the heart valve tissue of patients with Q fever endocarditis.
Typing Neisseria meningitidis by analysis of restriction fragment length polymorphisms in the gene encoding the class 1 outer membrane protein: application to assessment of epidemics throughout the last 4 decades in China
Authors: Zhu P , Hu X , Xu L
Published: January 2020

A typing method was developed for Neisseria meningitidis serogroup A by analysis of restriction fragment length polymorphisms (RFLP) of the class 1 outer membrane protein gene (porA). By using appropriate primers, an approximately 1,116-bp fragment of the porA gene was amplified by PCR and then was digested with the restriction endonuclease MspI. The digestion products were separated on 10% polyacrylamide gels and were stained with silver. One hundred three clinical isolates of group A N. meningitidis from 17 provinces of China collected over a 26-year period were analyzed. Results of MspI-generated RFLP profiles of PCR-amplified porA genes were compared with those obtained by conventional serosubtyping. There was a band of about 400 bp common to all strains examined, and the 103 strains of serogroup A resulted in 22 unique RFLP patterns. The differences in bands could be observed mainly in the range of 120 to 280 bp. The smaller fragments were useful in distinguishing meningococci with the same serosubtype. Three epidemic periods were characterized by the presence of three distinct genotypes (a1, a2, and a3), accounting for 74.5% of the strains examined (3.88, 26.21, and 44.66%, respectively). Three predominant RFLP patterns were correlated epidemiologically with cycles of epidemic meningococcal meningitis and were well-matched to the predominant serosubtypes (P1.9, P1.7, 10, and P1.9) that presented at the same prevalence cycles. The genotyping yielded information that allowed strains from one epidemic to be distinguished from those from another that would have been indistinguishable if only serotyping and serosubtyping were available. Therefore, the PCR-RFLP typing method was very useful in the epidemiologic investigation of group A meningococcal meningitis.
Comparison of GonoGen, GonoGen II, and MicroTrak direct fluorescent-antibody test with carbohydrate fermentation for confirmation of culture isolates of Neisseria gonorrhoeae
Authors: Kellogg J A , Orwig L K
Published: January 2020

When testing 248 clinical isolates of Neisseria gonorrhoeae, the sensitivity was 100% with GonoGen (Becton Dickinson Microbiology Systems), 99.6% (247 of 248) with GonoGen II (Becton Dickinson), 97.2% (241 of 248) with the MicroTrak direct fluorescent-antibody test (Syva), and 97.6% (242 of 248) with Rapid Fermentation Agar carbohydrates (Remel). Of 62 isolates of other Neisseria species, none was misidentified as N. gonorrhoeae by GonoGen, MicroTrak, or Rapid Fermentation Agar carbohydrates but 7 (31.8%) of 22 isolates of N. meningitidis gave strong, repeatedly false-positive results with GonoGen II. The sensitivity of all four assays was good to excellent, but all positive GonoGen II results should be confirmed with an independent assay, especially when isolates are recovered from sites where N. meningitidis is likely. Positive results from any of the assays should be routinely confirmed when dictated by specific clinical, legal, or microbiological circumstances.
Use of solid-phase immune electron microscopy for classification of Norwalk-like viruses into six antigenic groups from 10 outbreaks of gastroenteritis in the United States
Authors: Lewis D , Ando T , Humphrey C D , Monroe S S , Glass R I
Published: January 2020

Norwalk-like viruses observed in fecal specimens from 10 outbreaks of gastroenteritis investigated in the United States between 1987 and 1992 were analyzed by solid-phase immune electron microscopy. Outbreak virus strains were classified into six antigenic groups: the four types (UK1 to UK4) previously defined in the United Kingdom, Norwalk virus, and the Oklahoma agent that was newly defined in this study. The diversity of antigenic types demonstrated in these outbreaks was greater than previously recognized and will serve as a basis for characterization of these strains at the molecular level.
Risk of cross-colonization and infection by Pseudomonas aeruginosa in a holiday camp for cystic fibrosis patients
Authors: Hoogkamp-Korstanje J A , Meis J F , Kissing J , van der Laag J , Melchers W J
Published: January 2020

The risk of cross-colonization and subsequent infection by Pseudomonas aeruginosa in holiday camps for cystic fibrosis patients was studied in 91 children by culturing sputum at their arrival, at their departure, 2 months later, and at regular intervals thereafter. The isolated strains were subjected to serotyping, phage typing, pyocin typing, and genotyping by random amplified polymorphic DNA fingerprinting-PCR. It was concluded from random amplified polymorphic DNA fingerprinting-PCR typing that the Pseudomonas flora was not constant in most children. Some children harbored one genotype, whereas some harbored two or more different genotypes simultaneously. Most culture-positive children easily acquired a strain of another genotype which replaced the former one or coexisted with the original one. The incidence of sputum conversion was 7.7% in previously negative children; the incidence of permanent colonization and infection was 1.9%. This risk was comparable with that observed in the community. We conclude that the risk of cross-infection is trivial compared with the obvious joy and social benefit derived from a holiday camp.
Isolation of human immunodeficiency virus type 1 (HIV-1) RNA from feces by a simple method and difference between HIV-1 subpopulations in feces and serum
Authors: van der Hoek L , Boom R , Goudsmit J , Snijders F , Sol C J
Published: January 2020

A simple method for the isolation and subsequent detection of human immunodeficiency virus type 1 (HIV-1) RNA from feces is described. Viral RNA was isolated by the method developed by Boom et al. (R. Boom, C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990), which was adapted for feces. HIV-1 RNA was detected by reverse transcription (RT) followed by a nested PCR encompassing the V3 region. Reconstruction experiments revealed that the efficiencies of the extraction technique and the subsequent RT-PCR were not considerably affected by the varied composition of feces. The method was applied on fecal specimens from 18 HIV-1-infected individuals, among which were samples that had been stored for 9 years. It appeared that HIV-1 RNA was detectable in the feces of 12 persons (67%). Viral RNA was present in the feces of persons who fulfilled the criteria for CDC class II and CDC class III HIV infection as well as in patients who were diagnosed with AIDS (CDC class IV). Direct sequencing of amplimers obtained from paired fecal and serum specimens showed that differences in sequence heterogeneity existed. In one patient a remarkable difference in the HIV-1 sequences between isolates from feces and serum was observed. In conclusion, HIV-1 RNA is frequently present in the feces of HIV-1-infected individuals, and in some cases the HIV-1 subpopulation in feces differs from the HIV-1 subpopulation in serum.
Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species
Authors: Beutin L , Geier D , Zimmermann S , Karch H
Published: January 2020

Shiga-like toxin (verotoxin)-producing strains of Escherichia coli (SLTEC) originating from healthy cattle, sheep, goats, pigs, cats, and dogs were investigated for properties which are related to virulence of E. coli for humans. The slt-II (Shiga-like toxin II) and slt-IIc genes were frequent in SLTEC from healthy cattle and dogs but were rarely found in SLTEC from other animals. The slt-IIe gene was detected only in porcine SLTEC. SLTEC from goats and SLTEC from sheep were found to carry different SLT-II determinants which were not further characterized genetically. Sixty (28.8%) of 208 SLTEC from healthy animals showed diffuse adherence to HEp-2 cells. However, none of the strains was positive for genes specific for the local adherence (eaf), diffuse adherence (daa), or enteroaggregative (EAggEC) E. coli type. Only 3 (1.4%) of the 208 SLTEC were positive for attaching and effacing E. coli (eae) sequences. The enterohemolytic phenotype was present in 128 of the 208 SLTEC. Almost all enterohemolytic animal SLTEC were found to carry DNA sequences specific for the plasmid-encoded enterohemorrhagic E. coli hemolysin of E. coli O157. Bacteriophage-associated enterohemolysin (Ehly1 and Ehly2)-specific sequences were detected only in 14.4% of the 208 SLTEC and were linked with certain serotypes. The SLTEC from healthy animals constitute a very heterogeneous group of E. coli, and many of these strains appeared to be specific for their hosts. The absence of eae sequences in most animal SLTEC could indicate that these strains are less virulent for humans than the classical eae-positive enterohemorrhagic E. coli types.
Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients
Authors: Kulski J K , Khinsoe C , Pryce T , Christiansen K
Published: January 2020

The presence of mycobacteria in blood culture fluids (BACTEC) of AIDS patients with positive growth indices (GIs, > 20 U) was investigated by using a multiplex PCR to detect and identify members of the genus Mycobacterium, M. avium, M. intracellulare, and M. tuberculosis. Three different methods of extracting mycobacterial DNA from blood culture fluid were compared for use with the multiplex PCR. Mycobacterial cells were pelleted from a small aliquot of blood culture fluid by centrifugation, and the DNA was extracted from cells by heat lysis or a sodium iodide-isopropanol or a phenol-chloroform method. DNAs of different sizes were amplified from a region of the MPB70 gene of M. tuberculosis (372 bp) and from a region of the 16S rRNA gene of members of the genus Mycobacterium (1,030 bp), M. intracellulare (850 bp), or M. avium (180 bp) as a multiplex PCR in a single tube. The amplified DNA products were detected by agarose gel electrophoresis and ethidium bromide staining in all 41 (100%) positive cultures after sodium iodide-isopropanol extraction, in 18 (44%) after heat lysis, and in 5 (12%) after phenol-chloroform extraction. Of the 41 positive cultures, 38 were identified as M. avium and 2 were identified as M. intracellulare by both routine methods and multiplex PCR. The remaining mycobacterium was identified as M. intracellulare by routine methods and as M. avium by the multiplex PCR. Another six blood cultures that were negative for the presence of acid-fast bacilli after Ziehl-Neelson staining were also negative by PCR. The study shows that the multiplex PCR is a useful method for the detection and identification of either M. avium or M. intracellulare in small samples of cultured BACTEC 13A fluid with positive GIs ranging from 21 to 999 U. The average time to positive GI was 18 days (median, 13 days) and ranged between 8 and 42 days. The multiplex PCR may permit cultured mycobacteria to be identified at an earlier stage than the routine methods which have been adapted for use with the BACTEC system. The results also show that the method selected for extracting mycobacterial DNA from blood culture fluids is crucial for providing sensitive and accurate PCR results.
Random amplified polymorphic DNA and plasmid analyses used in investigation of an outbreak of multiresistant Klebsiella pneumoniae
Authors: Eisen D , Russell E G , Tymms M , Roper E J , Grayson M L , Turnidge J
Published: January 2020

Multiresistant Klebsiella pneumoniae strains with plasmid-borne extended-spectrum beta-lactamases (ESBL) are increasingly frequent nosocomial pathogens. A major outbreak of clinical infections, mainly involving patients in the Newborn Services Unit with limited spread to adult patients, occurred at our hospital. This epidemic was investigated by typing the isolates phenotypically and with random amplified polymorphic DNA analysis (RAPD) and plasmid analysis. Forty-eight isolates, consisting of 44 consecutive clinical isolates and 4 selected surveillance isolates, were studied. A single decamer primer was used for the RAPD, and this was effective in demonstrating that the majority of isolates (45 of 48) had the same profile. Three other isolates had different RAPD patterns identifying them as nonepidemic strains. Plasmids were extracted by alkaline lysis with Magic-miniprep kits from 10 isolates selected to represent the epidemic and nonepidemic strains. This method produced small (< 20-kb) plasmids; larger ESBL-carrying plasmids were not produced, but the small plasmids nonetheless allowed strain differentiation. Antibiotic susceptibility patterns alone were not reliable as strain indicators, since some isolates with the RAPD pattern characteristic of the epidemic strains did not express ESBL and therefore were susceptible to extended-spectrum cephalosporins. The investigation showed the predominance of a single epidemic strain that was transmitted between patients in the Newborn Services Unit. RAPD was the best of the methods used for detecting strain differences, and its speed and ability to type a wide variety of species suggest that it will be an increasingly useful molecular epidemiologic tool.
Evaluation of the monoclonal antibody-based kit Bengal SMART for rapid detection of Vibrio cholerae O139 synonym Bengal in stool samples
Authors: Qadri F , Hasan J A , Hossain J , Chowdhury A , Begum Y A , Azim T , Loomis L , Sack R B , Albert M J
Published: January 2020

A monoclonal antibody-based test, Bengal SMART, was developed for rapid detection of Vibrio cholerae O139 synonym Bengal directly from stool specimens. The test, which takes about 15 min to complete, was used to screen 189 diarrheal stool specimens. The results were compared with those of a monoclonal antibody-based coagglutination test (COAT) and the conventional culture methods used as the "gold standard" for detection of V. cholerae O139. The Bengal SMART test showed a sensitivity of 100% and a specificity of 97% in comparison with the gold standard. It also fared better than COAT, which had a sensitivity of 96% for rapid detection of V. cholerae O139 synonym Bengal. These results show that Bengal SMART is suitable for use in field settings for rapid diagnosis of cholera caused by V. cholerae O139.
Disseminated Microascus cirrosus infection in pediatric bone marrow transplant recipient
Authors: Krisher K K , Holdridge N B , Mustafa M M , Rinaldi M G , McGough D A
Published: January 2020

Microascus cirrosus Curzi and its associated anamorphic state, Scopulariopsis, were recovered from the cutaneous lesion of a 12-year-old male who had undergone an autologous bone marrow transplantation for acute myelogenous leukemia. Histopathology sections from the biopsied lesion demonstrated septate hyphae consistent with a fungal etiology. Radiographic studies of the lungs subsequent to progression of the lesion revealed a consolidation in the right upper lobe suggesting a primary focus of infection. While M. cirrosus is fairly abundant in nature and widely distributed in stored grains in North America, this is, to our knowledge, the first reported human infection by this species. Salient characteristics for the identification of this dematiaceous ascomycete, M. cirrosus, will be presented.
Sensitivity of sandwich enzyme-linked immunosorbent assay for Cryptococcus neoformans polysaccharide antigen is dependent on the isotypes of the capture and detection antibodies
Authors: Mukherjee S , Casadevall A
Published: January 2020

Immunoglobulin M (IgM) and IgG1 monoclonal antibody isotype switch variants to Cryptococcus neoformans capsular polysaccharide were used to study the sensitivity of a double sandwich enzyme-linked immunosorbent assay (ELISA). The most sensitive ELISA configurations used IgG1 monoclonal antibody absorbed on polystyrene plates or IgM immobilized with goat antisera for antigen capture.
Stability of karyotype in serial isolates of Candida albicans from neutropenic patients
Authors: Barton R C , van Belkum A , Scherer S
Published: January 2020

Serial isolates of Candida albicans were obtained from 20 patients with leukemia over periods of up to 8 months. The fingerprinting of these isolates by interrepeat PCR and random amplified polymorphic DNA PCR has been described previously (A. van Belkum, W. Melchers, B. E. de Pauw, S. Scherer, W. Quint, and J. F. Meis, J. Infect. Dis. 169:1062-1070, 1994). Contour-clamped homogeneous electric field gel electrophoresis was used to examine the chromosomes of these isolates. When changes in the karyotype were seen in a series of isolates, additional interrepeat PCR and Southern blotting with a repeat DNA sequence from the 27A family were performed. These two genotyping tools were used to determine if karyotypic changes seen in a series of isolates were due to chromosome rearrangements in a single strain or due to colonization with more than one strain. It was determined that changes in karyotype in a series of strains indicated infection by a new strain.
Evaluation of five new plating media for isolation of Salmonella species
Authors: Dusch H , Altwegg M
Published: January 2020

A three-phase study was conducted to compare Hektoen enteric agar (HE), Rambach agar (Ra), SM-ID medium (SM), xylose-lysine-Tergitol 4 agar (XLT4), novobiocin-brilliant green-glycerol-lactose agar (NBGL), and modified semisolid Rappaport-Vassiliadis medium (MSRV) for the recovery of nontyphoid salmonellae from stool specimens. After evaluation of the first two phases, which resulted in the elimination of Ra, SM, and NBGL, 593 consecutive stool samples were investigated by plating them directly and after tetrathionate enrichment at 37 degrees C on HE, XLT4, and MSRV. A total of 82 Salmonella-positive stool specimens were detected (positivity rate, 13.8%). Sensitivities for direct plating and after tetrathionate enrichment were 32.9 and 86.6%, respectively, for XLT4, 63.4 and 100.0%, respectively, for MSRV, and 34.1 and 79.3%, respectively, for HE. Specificities (percentage of morphologically suspicious colonies that were indeed salmonellae) were 100.0 and 99.8%, respectively, for XLT4, 99.0 and 98.8%, respectively, for MSRV, and 67.9 and 75.0%, respectively, for HE. The use of MSRV instead of HE increased the isolation rate of salmonellae by 26.2% (65 versus 82 strains isolated from HE and MSRV, respectively). We conclude that MSRV is the most sensitive medium tested and is a very specific medium for the isolation of nontyphoid salmonellae from stool specimens. However, its semisolid nature is a disadvantage and requires careful handling in the laboratory, especially when salmonellae are present. XLT4 had a sensitivity comparable to that of HE and a nearly 100% specificity and can be regarded as an alternative for the isolation of nontyphoid salmonellae from stool samples.
Comparison of commercial enzyme immunoassay kits with plaque reduction neutralization test for detection of measles virus antibody
Authors: Ratnam S , Gadag V , West R , Burris J , Oates E , Stead F , Bouilianne N
Published: January 2020

Four commercially available enzyme immunoassay (EIA) kits were evaluated in comparison with the plaque reduction neutralization (PRN) test for detection of measles virus antibody. The EIA kits, Enzygnost (Behring), Diamedix, Vidas (bioMerieux Vitek), and Measlestat (Biowhittaker), were assessed with two PRN cutoff titers: a PRN titer of 8, the lowest detectable antibody level by the PRN test under the test conditions, and a titer of 120, which has been shown to be the minimum protective antibody titer. At a PRN cutoff titer of 8, the sensitivity was 88.2, 91.1, 74.6, and 69.8% for Behring, Diamedix, Vidas, and Biowhittaker EIA tests, respectively, with negative predictive values ranging from 22.7 to 45.5%. The specificity was 93.8% for Diamedix and 100% for the rest. At a PRN cutoff titer of 120, the sensitivity and specificity, respectively, were 100 and 90.7% (Behring), 98.2 and 58.8% (Diamedix), 90.6 and 94.5% (Vidas), and 85.7 and 96.4% (Biowhittaker). At this PRN cutoff titer, the negative predictive values of all EIA tests improved considerably, ranging from 70.7 to 100%. The EIA results showed an excellent association with PRN results when the PRN titers of the test samples were either < 8 or > 1,052. Discrepancies occurred especially when testing samples having PRN titers in the range of 8 to 120, indicating lack of sensitivity of the EIA tests in detecting measles virus antibody at low levels. Maternally derived measles virus antibody at this level has been shown to interfere with measles vaccine response in children and hence has implications from the standpoint of measles immunization. The ready availability, ease of operation, and rapid turnaround time are strong plus points of the EIA kits, and they could be useful in a clinical laboratory setting for routine application, but they may have limited use in vaccine-related studies and seroepidemiological surveys.
Molecular subtyping scheme for serotypes HS1 and HS4 of Campylobacter jejuni
Authors: Owen R J , Sutherland K , Fitzgerald C , Gibson J , Borman P , Stanley J
Published: January 2020

We describe a molecular subtyping scheme for two principal O (heat-stable [HS]) serotypes of Campylobacter jejuni, HS1 and the HS4 complex. A 16S rRNA gene-specific probe confirmed that almost all the C. jejuni strains had three copies of this gene, and strains could be assigned with complete typeability to 1 of 16 combined (Pst1 and HaeIII) 16S ribotypes. Macrorestriction profiles (mrps) consisting of up to 10 SmaI fragments from approximately 40 to approximately 480 kbp were resolved by pulsed-field gel electrophoresis (PFGE). There were 11 mrps among the HS1 strains and 9 mrps among HS4 strains which corresponded to valid types--they occurred in multiple isolates, hosts, places, and times. There were 14 additional single-strain mrp fingerprints in HS1 and 20 in HS4. PFGE exhibited complete typeability when formaldehyde fixation of cells was employed, and PFGE was generally more differential than ribotyping. The data presented elucidate a high-resolution genotypic subtyping scheme for these common subspecific phenotypes of C. jejuni, which is both coherent and efficient for epidemiological purposes.
Simple determination of human immunodeficiency virus type 1 syncytium-inducing V3 genotype by PCR
Authors: Fouchier R A , Brouwer M , Broersen S M , Schuitemaker H
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) phenotype variability plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected individuals is associated with a rapid decline of CD4+ T cells, rapid disease progression, and reduced survival time after AIDS diagnosis. The strong association between the SI capacity of HIV-1 and the presence of positively charged amino acid residues at positions 306 and/or 320 in the third variable domain (V3) of gp120 could here be confirmed in 97% of 402 primary HIV-1 isolates, indicating that the V3 genotype may be useful for prediction of the viral phenotype. The V3 DNA sequences revealed a remarkably limited codon usage for the amino acid residues that are responsible for virus phenotype. On the basis of this limited SI-specific DNA sequence variation, four SI-specific oligonucleotides were designed for selective amplification of V3 from SI but not non-SI HIV-1 isolates. This PCR analysis allowed the prediction of the biological phenotype of HIV-1 isolates on the basis of the V3 genotype and may prove to be useful for monitoring SI capacity of HIV-1 isolates in infected individuals.
Epidemiological typing of Stenotrophomonas (Xanthomonas) maltophilia by PCR
Authors: Chatelut M , Dournes J L , Chabanon G , Marty N
Published: January 2020

We used two PCR methods for epidemiological typing of Stenotrophomonas (Xanthomonas) maltophilia with either arbitrary primers (random amplified polymorphic DNA) or enterobacterial repetitive intergenic consensus sequences as primers (ERIC-PCR). The analysis was performed with 38 isolates of S. maltophilia, comprising 9 nosocomial isolates from a burn unit, 20 other clinical isolates epidemiologically unrelated, and 9 isolates from one cystic fibrosis patient. Both methods indicated that all of the nosocomial episodes were independent. In contrast, the nine isolates from the cystic fibrosis patient were assigned to very closely related profiles, especially by ERIC-PCR. We conclude that random amplified polymorphic DNA and ERIC-PCR have comparable reproducible and discriminatory powers for epidemiological typing of S. maltophilia, but ERIC-PCR profiles can be more easily evaluated.
Specific antibody detection in invasive aspergillosis by analytical isoelectrofocusing and immunoblotting methods
Authors: Hearn V M , Pinel C , Blachier S , Ambroise-Thomas P , Grillot R
Published: January 2020

Aspergillus fumigatus antigens have been tested to determine their potential as aids in the diagnosis of invasive aspergillosis (IA). Immunoglobulin G (IgG) antibodies to these antigens were detected by analytical isoelectrofocusing in conjunction with immunoblotting. A total of 12 antigenic fractions, including culture filtrates and surface and mycelial extracts of A. fumigatus, were investigated. Eleven were reactive with serum specimens from patients with aspergilloma, which served as positive controls for the evaluation of a specific IgG response. Eight of 12 antigens showed good responses with serum specimens from patients with allergic bronchopulmonary aspergillosis, which were used to assess the sensitivity of IgG detection. No measurable reactivity was detected in 18 negative control serum specimens, while 11 of 13 patients with proven, highly probable, or probable cases of IA had anti-Aspergillus IgG to multiple antigenic preparations. Patients with IA who were capable of mounting a substantial humoral response to Aspergillus antigens gave an antibody profile with five antigenic preparations which seemed to be characteristic of the disease. Data show that this method is highly sensitive and may allow the selection of fractions which are both highly antigenic and specific for the detection of antibodies to Aspergillus antigens. They also indicate that the use of a spectrum of antigenic molecules is advisable, given the variability observed in the immune responses of individual patients.
Characterization of the bovine group A rotavirus strain neonatal calf diarrhea virus-Cody (NCDV-Cody)
Authors: Lu W , Duhamel G E , Hoshino Y , Benfield D A , Nelson E A , Hesse R A
Published: January 2020

The neonatal calf diarrhea virus-Cody (NCDV-Cody) strain was found to contain a mixture of rotaviruses with G6 and G8 VP7 genes. Challenge exposure of calves with the mixed virus inoculum indicated that both viruses were maintained by passage in vivo. This is the first P1:G8 rotavirus to be characterized in cattle in the United States.
Rapid and economical method for species identification of clinically significant coagulase-negative staphylococci
Authors: Ieven M , Verhoeven J , Pattyn S R , Goossens H
Published: January 2020

Four methods for the species identification of coagulase-negative staphylococci in the medical microbiology laboratory were compared with 444 consecutive isolates. The methods included (i) the reference method based on growth tests, (ii) API ID 32 Staph (bioMérieux), (iii) Staph-Zym (Rosco), and (iv) a rapid 4-h method developed in our laboratory (UZA method). The last method is based on the detection within 4 h of enzymatic activity of heavy bacterial suspensions in three substrate solutions (nongrowth tests). For 16.5% of the isolates some supplementary growth tests read after 24 h had to be added to the enzyme data for satisfactory identification. The reference method failed to identify four isolates. Of the 440 isolates identified by the reference method, API ID 32 Staph, Staph-Zym, and the UZA method correctly identified 419 (95.2%), 429 (97.5%), and 430 (97.7%) and misidentified 8 (1.8%), 4 (0.9%), and 1 (0.2%), respectively. Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis, S. schleiferi, and S. capitis were identified with an accuracy of 98 to 100% by all the systems tested. S. capitis subsp. ureolyticus was not recognized by the API ID 32 system because the biochemical profiles for it are not yet included in the corresponding database. Whereas API ID 32 identified all 13 S. warneri isolates, both Staph-Zym and the UZA method missed 2 of these. S. hominis was identified with the least accuracy by the API ID 32 system (26 of 39 isolates), whereas the UZA and Staph-Zym methods identified 36 of the isolates belonging to this species. The UZA method did not identify any of the S. cohnii, S. xylosus, S. lentus, and S. sciuri strains, since it included no discriminatory tests for these species, because they are extremely rarely found in humans. Of all 440 isolates tested, the UZA method failed to identify 9 and misidentified 1 other. Eighty-one percent of the isolates were identified within 4 h and 97.7% were identified after 24 h, at considerably less expense than by the API ID 32 Staph and Staph-Zym methods.