bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique oligonucleotide primers in PCR for identification of Cryptococcus neoformans
Authors: Mitchell T G , Freedman E Z , White T J , Taylor J W
Published: January 2020

On the basis of a comparison of rRNA sequences coding the internal transcribed spacer (ITS) and 5.8S regions of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans, and members of its most closely related taxa, unique oligonucleotides were designed for the specific amplification of DNA only from C. neoformans. Various combinations of these primers, designated CN4, CN5, and CN6, and the previously described fungal primers ITS1 and ITS2 were tested in PCR for their ability to amplify DNA from 37 strains of C. neoformans and 31 other isolates representing 18 species of yeasts. The combination of primers CN4 and CN5 amplified DNA from both varieties of C. neoformans but from none of the other species tested. Other pairs of primers (namely, CN5-CN6, CN4-ITS1, and CN6-ITS1) amplified DNA only from C. neoformans and from the saprophyte Filobasidiella depauperata, which is the only other member of the genus Filobasidiella. With appropriate controls, these specific oligonucleotides may be used as primers in PCR to identify C. neoformans.
Comparison of rapid detection methods for influenza A virus and their value in health-care management of institutionalized geriatric patients
Authors: Leonardi G P , Leib H , Birkhead G S , Smith C , Costello P , Conron W
Published: January 2020

Respiratory specimens from 160 geriatric patients with suspected influenza illness were used to evaluate the abilities of two enzyme immunoassays (EIAs; Directigen FLU-A [Becton Dickinson Microbiology Systems, Cockeysville, Md.] and Prima EIA [Baxter/Bartels Diagnostics, Inc., Issaquah, Wash.]) and direct immunofluorescence testing (immunofluorescence assay [IFA]) to identify influenza A virus. In comparison with culture isolation, the sensitivities and specificities of the IFA, Directigen FLU-A, and Prima EIA were 92.5 and 97.2%, 86.8 and 99.1%, and 92.5 and 98.1%, respectively. In contrast to EIA, IFA was labor intensive and required a high degree of technical expertise, and the results of IFA were difficult to interpret. These factors may preclude the use of IFA for testing large numbers of specimens. A retrospective epidemiologic survey of influenza infection was done in six geriatric institutions which had used either rapid and culture testing or culture alone. Preventable cases of influenza A virus infection ranged from 9 to 38% of all cases in facilities which used culture testing only and which had not instituted amantadine prophylaxis. The use of direct specimen testing is recommended as an adjunct to culture isolation for the identification of influenza A virus. Use of a combination of these methods permits the timely administration of appropriate antiviral therapy and infection control measures, while it also permits the antigenic surveillance of circulating influenza strains, which is necessary for present vaccine efficacy evaluations and the creation of future effective vaccine formulations.
Plasmid DNA fingerprinting of Acinetobacter species other than Acinetobacter baumannii
Authors: Seifert H , Schulze A , Baginski R , Pulverer G
Published: January 2020

During the last years Acinetobacter species have emerged as clinically significant pathogens. Most infections are nosocomially acquired and mainly due to Acinetobacter baumannii. Little is known about the epidemiology and clinical significance of unnamed Acinetobacter species 3 (the second most often encountered member of the genus Acinetobacter) and other Acinetobacter species such as A. johnsonii, A. junii, and A. lwoffii. Seventy-five clinical isolates of Acinetobacter species other than A. baumannii (Acinetobacter species 3, n = 37; A. johnsonii, n = 20; A. junii, n = 8; A. lwoffii, n = 10) recovered from 66 patients over a period of 12 months were analyzed by plasmid DNA fingerprinting. Plasmids were found in 84.4% of Acinetobacter species 3 isolates and in all A. johnsonii, A. junii, and A. lwoffii isolates. Strains harbored up to 15 plasmids each. Almost every isolate gave a unique plasmid pattern. With one exception, identical plasmid profiles were detected only in corresponding isolates recovered from blood cultures and intravascular catheters from a given patient. Plasmid DNA fingerprinting proved to be useful for typing Acinetobacter species other than A. baumannii. There was no evidence of patient-to-patient transmission or hospital outbreaks due to these species. This finding is in contrast to the results obtained in studies of the hospital epidemiology of A. baumannii.
Interlaboratory agreement study of a double set of PCR plasmid primers for detection of Chlamydia trachomatis in a variety of genitourinary specimens
Authors: Mahony J B , Luinstra K E , Waner J , McNab G , Hobranzska H , Gregson D , Sellors J W , Chernesky M A
Published: January 2020

We conducted a tricenter interlaboratory agreement study to assess the agreement of PCR results obtained for detection of Chlamydia trachomatis in genitourinary specimens. A total of 120 specimens (49 positive and 71 negative), including 20 first-void urine samples, 50 endocervical and 50 urethral swabs (40 males), were coded and sent from a reference laboratory (laboratory A) to two other laboratories. Laboratories B and C were provided with a standardized protocol and reagent package including two sets of plasmid PCR primers (KL1-KL2 and T1-T2) and were asked to test each specimen with the first set of primers (KL1-KL2) and to confirm positives with the second set of primers (T1-T2). Laboratory B identified 47 of 49 positives and 69 of 70 negatives (one specimen dried up on shipping) following the initial PCR, for an accuracy of 97.5% (116 of 119), and 47 of 49 positives and 70 of 70 negatives after confirmatory testing of the positives, for an accuracy of 98.3% (117 of 119). Laboratory C identified 42 of 49 positives and 70 of 70 negatives for the initial PCR, for an accuracy of 94.1% (112 of 119), and 39 of 42 positives and 70 of 70 negatives for the confirmatory PCR, for an accuracy of 91.6% (109 of 119). The overall accuracy of PCR testing was 96.6% (345 of 357). The kappa agreement statistics for agreement between pairs of laboratories after confirmation of positives were 0.97 for laboratories A and B, 0.83 for laboratories B and C, and 0.83 for laboratories A and C. Use of the confirmatory PCR improved the specificity and overall accuracy of results for individual laboratories but reduced slightly the results obtained for agreement between laboratories. These results demonstrate that when standardized reagents and protocols are used, PCR results are highly reproducible and excellent agreement between laboratories is obtainable.
Detection of bovine trichomoniasis with a specific DNA probe and PCR amplification system
Authors: Ho M S , Conrad P A , Conrad P J , LeFebvre R B , Perez E , BonDurant R H
Published: January 2020

Trichomoniasis is a widespread, economically important venereal disease of cattle which causes infertility and abortion. Effective control of trichomoniasis has been impeded by the insensitivity of traditional diagnostic procedures, which require the isolation and cultivation of the parasite, Tritrichomonas foetus, from infected cattle. We developed a 0.85-kb T. foetus DNA probe by identifying conserved sequences in DNAs from T. foetus that were isolated from cattle in California, Idaho, Nevada, and Costa Rica. The probe hybridized specifically to DNAs of T. foetus isolates from different geographic areas but not to DNA preparations of Trichomonas vaginalis, bovine cells, or a variety of bacteria from cattle. The probe detected DNA from a minimum of 10(5) T. foetus organisms. To improve sensitivity, a partial sequence of the probe was used to identify oligonucleotide primers (TF1 and TF2) which could be used to amplify a 162-bp product from T. foetus DNAs by PCR. A chemiluminescent internal T. foetus sequence probe was hybridized to Southern blots of the amplification product. This system detected as few as one T. foetus organism in culture media or 10 parasites in samples containing bovine preputial smegma. Analysis of 52 clinical samples showed that 47 (90.4%) of the 52 samples were correctly identified, with no false-positive reactions. In comparison, the traditional cultivation method detected 44 (84.6%) of the 52 samples from T. foetus-infected and uninfected bulls. These results indicate that the PCR-based amplification system could be a useful alternative method for the diagnosis of bovine trichomoniasis.
Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis
Authors: Camargo Z P , Gesztesi J L , Saraiva E C , Taborda C P , Vicentini A P , Lopes J D
Published: January 2020

Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM.
Development of interpretive criteria and quality control limits for broth microdilution and disk diffusion antimicrobial susceptibility testing of Streptococcus pneumoniae
Authors: Jorgensen J H , Swenson J M , Tenover F C , Ferraro M J , Hindler J A , Murray P R
Published: January 2020

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.
Comparative study of broth macrodilution and microdilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards' proposed standard
Authors: Barchiesi F , Colombo A L , McGough D A , Rinaldi M G
Published: January 2020

A comparative study of broth macro- and microdilution methods for susceptibility testing of fluconazole, itraconazole, flucytosine, and amphotericin B was conducted with 273 yeasts. The clinical isolates included 100 Candida albicans, 28 Candida tropicalis, 25 Candida parapsilosis, 15 Candida lusitaniae, 15 Candida krusei, 50 Cryptococcus neoformans var. neoformans, 25 Torulopsis (Candida) glabrata, and 15 Trichosporon beigelii strains. Both methods were performed according to the National Committee for Clinical Laboratory Standards' (NCCLS) recommendations (document M27-P). For fluconazole, itraconazole, and flucytosine, the endpoint was the tube that showed 80% growth inhibition compared with the growth control for the macrodilution method and the well with slightly hazy turbidity (score 1) compared with the growth control for the microdilution method. For amphotericin B, the endpoint was the tube and/or well in which there was absence of growth. For the reference macrodilution method, the MICs were determined after 48 h of incubation for Candida spp., T. glabrata, and T. beigelii and after 72 h for C. neoformans var. neoformans. For the microdilution method, either the first-day MICs (24 h for all isolates other than C. neoformans and 48 h for C. neoformans var. neoformans) or the second-day MICs (48 and 72 h, respectively) were evaluated. The agreement within one doubling dilution of the macrodilution reference for all drugs was higher with the second-day MICs than with the first-day MICs for the microdilution test for most of the tested strains. General agreement was 92% for fluconazole, 85.7% for itraconazole, 98.3% for flucytosine, and 96.4% for amphotericin B. For C. neoformans var. neoformans and T. beigelii, the agreement of the first-day reading was higher than that of the second-day reading for fluconazole (94 versus 92%, respectively, for C. neoformans var. neoformans, and 86.7 versus 80%, respectively, for T. beigelii). Our studies indicate that the microdilution technique performed following the NCCLS guidelines with a second-day reading is a valid alternative method for testing fluconazole, itraconazole, flucytosine, and amphotericin B against these eight species of yeasts.
Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens
Authors: Kokai-Kun J F , Songer J G , Czeczulin J R , Chen F , McClane B A
Published: January 2020

Clostridium perfringens enterotoxin (CPE) is an important sporulation-associated virulence factor in several illnesses of humans and domestic animals, including C. perfringens type A food poisoning. Therefore, the ability to determine the enterotoxigenicity of food or fecal C. perfringens isolates with simple, rapid assays should be helpful for epidemiologic investigations. In this study, Western immunoblotting (to detect CPE production in vitro) was compared with PCR assays and digoxigenin-labeled probe assays (to detect all or part of the cpe gene) as a method for determining the enterotoxigenicity of C. perfringens isolates. The cpe detection assays yielded reliable results with DNA purified from vegetative C. perfringens cultures, while Western immunoblots required in vitro sporulation of C. perfringens isolates to detect CPE production. Several cpe-positive C. perfringens isolates from diarrheic animals did not sporulate in vitro under commonly used sporulation-inducing conditions and consequently tested CPE negative. This result indicates that cpe gene detection and serologic CPE assays do not necessarily yield similar conclusions about the enterotoxigenicity of a C. perfringens isolate. Until further studies resolve whether these cpe-positive isolates which do not sporulate in vitro can or cannot sporulate and produce CPE in vivo, it may be preferable to use cpe detection assays for evaluating C. perfringens isolate enterotoxigenicity and thereby avoid potential false-negative conclusions which may occur with serologic assays.
Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix
Authors: Schachter J , Stamm W E , Quinn T C , Andrews W W , Burczak J D , Lee H H
Published: January 2020

We performed a multicenter evaluation of ligase chain reaction (LCR) in the diagnosis of Chlamydia trachomatis infection of the cervix. This LCR provides an amplification of target sequences within the chlamydial cryptic plasmid. The LCR results were compared with those of isolation in cell culture. Discrepant (tissue culture-negative and LCR-positive) test results were resolved by the application of a direct immunofluorescent-antibody test to detect chlamydial elementary bodies and by the use of alternate DNA primers that targeted the chlamydial major outer membrane protein gene. A total of 234 of 2,132 specimens (10.9%) could be confirmed as containing C. trachomatis. Of these, 152 were detected by isolation in cell culture and 221 were detected by LCR. The corresponding sensitivities were 94% for LCR and 65% for cell culture. There was greater variability among study site results for cell culture sensitivity (52 to 92%) than for LCR sensitivity (87 to 98%). The specificity of each test was greater than 99.9%. Thus, LCR offers a highly sensitive nonculture method for detecting chlamydial infection of the cervix.
Detection of Campylobacter upsaliensis from a blood culture by using the BacT/Alert system
Authors: Carnahan A M , Beadling J , Watsky D , Ford N
Published: January 2020

Campylobacter upsaliensis was isolated from the blood of a 60-year-old female with hairy cell leukemia. This spiral-shaped organism was detected in the aerobic BacT/Alert bottle (Organon Teknika, Durham, N.C.) by acridine orange staining and was recovered only on chocolate agar in a microaerophilic atmosphere at 35 degrees C.
Reverse transcriptase-PCR assay for detection of hog cholera virus
Authors: Harding M , Lutze-Wallace C , Prud'Homme I , Zhong X , Rola J
Published: January 2020

A reverse transcriptase-PCR strategy was developed for the detection of hog cholera virus. Hog cholera virus template was amplified from tissue culture fluids and from tissues and blood of infected pigs, but not from samples containing other pestiviruses. Restriction endonuclease analysis identified samples as historic or recent isolates.
Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR
Authors: Maass M , Dalhoff K
Published: January 2020

Amplification inhibitors can lead to false-negative results for PCR. In order to evaluate the reliability of PCR for the detection of Chlamydia pneumoniae, the presence of PCR inhibitors in 75 bronchoalveolar lavage specimens was assessed after treatment by various sample preparation methods. Specimens were collected from patients with acute respiratory infections, including four cases of proven C. pneumoniae infection. Substances inhibitory to the amplification of chlamydial DNA continued to be present in 12% of the samples treated according to the commonly used single-step proteinase K digestion and in 31% of the samples processed by heat treatment. However, the complexing of DNA-contaminating proteins and polysaccharides from digested specimens to cetyltrimethylammonium bromide (CTAB) followed by DNA extraction efficiently removed inhibitors from all experimental samples and provided subsequent identification of all positive clinical samples by PCR. The CTAB method and proteinase K treatment had comparable detection limits of approximately 0.01 inclusion-forming units. CTAB-based DNA purification of respiratory specimens is recommended to increase the diagnostic sensitivity of PCR and confidence in negative results.
Discriminatory genomic fingerprinting of Legionella pneumophila by pulsed-field electrophoresis
Authors: Johnson W M , Bernard K , Marrie T J , Tyler S D
Published: January 2020

Eight strains of Legionella pneumophila were used to optimize cleavage of DNA with BssHII, SalI, or SpeI and separation by pulsed-field electrophoresis. Isolates from a community outbreak involving a contaminated hot tub were genomically identical. Cleavage patterns were distinctly different for unrelated environmental and nosocomial strains from a single hospital.
Risk factors for acquiring ampicillin-resistant enterococci and clinical outcomes at a Canadian tertiary-care hospital
Authors: McCarthy A E , Victor G , Ramotar K , Toye B
Published: January 2020

The number of ampicillin-resistant enterococci (ARE) was noted to be increased at our teaching hospital. To determine the risk factors for acquiring this organism and to compare clinical outcomes, over a 5-month period 38 patients infected or colonized with ARE were compared with 76 patients, infected or colonized with ampicillin-susceptible enterococci (ASE). Risk factors included nosocomial acquisition, duration of hospitalization, admission to a medical service, prior antimicrobial therapy, and combination therapy for at least 7 days. The mortality rate of patients infected or colonized with ARE was higher than that of patients infected or colonized with ASE (34 versus 14%; P = 0.03), but most deaths did not appear to be related to enterococcal infection. Over a 2-year period, 16 patients with ARE bacteremia were also compared with 23 patients with ASE bacteremia. The risk factors associated with ARE bacteremia also included nosocomial acquisition, duration of hospitalization, and prior antimicrobial therapy. The mortality of patients with ARE bacteremia was also higher than that of patients with ASE bacteremia (81 versus 30%; P = 0.003), with most deaths being due to the underlying disease or a complication of it. Typing of ARE isolates by pulsed-field gel electrophoresis showed that two genotypes predominated in our institution. A prolonged hospital stay, exposure to multiple antimicrobial agents, and perhaps nosocomial transmission are important factors in acquiring ARE. The presence of ARE may also be a marker for poor outcome.
Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction
Authors: Chernesky M A , Jang D , Lee H , Burczak J D , Hu H , Sellors J , Tomazic-Allen S J , Mahony J B
Published: January 2020

From April to September 1993, 305 men and 447 women in Hamilton, Canada, consented to the collection of a urethral or cervical swab, respectively, for culture and 20 ml of first-void urine (FVU) for testing by the enzyme immunoassay Chlamydiazyme and by ligase chain reaction (LCR) in the form of a kit from Abbott Laboratories called LCx Chlamydia trachomatis. Evaluation of test performance with each specimen was calculated on the basis of an expanded "gold standard" of a patient found to be positive by culture or by a confirmed nonculture test. By using this expanded standard, the prevalence of infection was determined to be 6% (27/447) for the women and 18.4% (56/305) for the men. LCR testing of FVU in both studies was the most sensitive approach (96%). The performance of Chlamydiazyme was as follows: cervical swab, 78.3% sensitivity; female FVU, 37% sensitivity; and male FVU, 67.9% sensitivity. Culture was the least sensitive approach to diagnosis: female cervix, 55.6%; and male urethra, 37.5%. LCR testing of FVU from men or women diagnosed the greatest number of genitourinary tract infections with no false positives.
Ribotyping as an additional molecular marker for studying Neisseria meningitidis serogroup B epidemic strains
Authors: Tondella M L , Sacchi C T , Neves B C
Published: January 2020

The molecular method of ribotyping was used as an additional epidemiological marker to study the epidemic strains of Neisseria meningitidis serogroup B, referred to as the ET-5 complex, responsible for the epidemic which occurred in greater São Paulo, Brazil. Ribotyping analysis of these strains showed only a single rRNA gene restriction pattern (Rb1), obtained with ClaI restriction enzyme. This method, as well as multilocus enzyme electrophoresis, provided useful information about the clonal characteristics of the N. meningitidis serogroup B strains isolated during this epidemic. The N. meningitidis serogroup B isolates obtained from epidemics which occurred in Norway, Chile, and Cuba also demonstrated the same pattern (Rb1). Ribotyping was a procedure which could be applied to a large number of isolates and was felt to be appropriate for routine use in laboratories, especially because of the convenience of using nonradioactive probes.
Cryptosporidium antigen detection in human feces by reverse passive hemagglutination assay
Authors: Farrington M , Winters S , Walker C , Miller R , Rubenstein D
Published: January 2020

A reverse passive hemagglutination (RPH) assay was developed for Cryptosporidium oocyst antigen with an antioocyst monoclonal antibody (MAb; MAb-C1) coupled to stabilized sheep erythrocytes. RPH was compared with microscopy of auramine-phenol-stained smears of 56 oocyst-positive fecal samples, each of which was tested blindly by RPH with two oocyst-negative samples received on the same day (a total of 112 controls). Thirty-nine additional fecal samples from human immunodeficiency virus type 1 antibody-positive patients with diarrhea (10 of which were positive in auramine-phenol-stained smears) were stored at -20 degrees C before testing. Thirty specimens with a variety of other fecal pathogens (all negative for oocysts) were also tested. Of the 237 samples tested, 69 were positive by one or both methods: 65 by RPH and 66 by microscopy. The kappa coefficient of agreement between the methods was very high at 0.926. The sensitivity of RPH was 93.9%, the specificity was 98.2%, the positive predictive value was 95.4%, and the negative predictive value was 97.7%. Visible oocyst numbers and RPH titers were measured after storage of fecal samples and oocyst concentrates for 8 days at 4 degrees C. Oocyst morphology was generally poor in specimens from the human immunodeficiency virus type 1 antibody-positive group, and it degenerated during the 8-day storage experiments. MAb-C1-reactive antigen eluted from oocysts to give progressively higher reciprocal titers during storage, and it was partially removed from the oocysts by concentration. RPH is a promising technique for the detection of Cryptosporidium antigen in human feces and may be useful when specimens are stored before testing. Studies of the sensitivity of Cryptosporidium immunoassays should take into account the possible release of antigen from oocysts.
Osteomyelitis of the sternum caused by Apophysomyces elegans
Authors: Eaton M E , Padhye A A , Schwartz D A , Steinberg J P
Published: January 2020

Apophysomyces elegans, a member of the family Mucoraceae, was found to infect the chest wall and sternum of an immunocompetent man following minor trauma. As in previous cases, amphotericin B therapy alone was inadequate. Extensive surgical debridement was required in order to eradicate the infection.
Effects of volume and periodicity on blood cultures
Authors: Li J , Plorde J J , Carlson L G
Published: January 2020

Blood specimens collected for culture by using the high-volume resin-based BACTEC system over an 18-month period at the Seattle Veterans Administration Center were examined in this study. Of 7,783 cultures obtained, 624 were classified as true positives. Patients in this group had between 20 and 60 ml of blood drawn per culture and separated into 10-ml aliquots for incubation. Analysis of the results stratified by cultured volume and time interval between specimen collection accorded yield advantage to culture volume at the maximal amounts tested. No advantage was observed with any particular interval of collection. Increasing cultured volume from 20 to 40 ml increased yield by 19%. Increasing cultured volume from 40 to 60 ml increased yield by an additional 10%. The same effect was seen whether cultures were drawn simultaneously or serially within 24 h. These observations support other reports demonstrating increased yield with increased cultured blood volume. However, they demonstrate increases in yield at volumes much higher than previously considered. In conclusion, this study demonstrates that high-volume blood cultures drawn serially or simultaneously return the best yields.
Detection of human immunodeficiency virus type 1 (HIV-1) in heel prick blood on filter paper from children born to HIV-1-seropositive mothers
Authors: Nyambi P N , Fransen K , De Beenhouwer H , Chomba E N , Temmerman M , Ndinya-Achola J O , Piot P , van der Groen G
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) DNA PCR results of 94 dried blood spot (DBS) samples on filter paper and corresponding venous blood in EDTA obtained from infants born to HIV-1-seropositive mothers were compared. In addition, the results of HIV-1 DNA PCR on DBS and the HIV-1 RNA PCR from plasma of 70 paired samples were compared. A 100% specificity and a 95% sensitivity for HIV-1 DNA PCR on DBS compared with results for venous blood were observed for the 94 paired samples. The results of the DBS HIV-1 DNA PCR and HIV-1 RNA PCR of 70 corresponding plasma samples correlated perfectly (100%). The DBS HIV-1 DNA PCR method proved reliable for HIV-1 detection.
Typing of Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting
Authors: Ursi D , Ieven M , van Bever H , Quint W , Niesters H G , Goossens H
Published: January 2020

PCR fingerprinting was used to characterize clinical isolates of Mycoplasma pneumoniae. Among 24 strains tested, two types were distinguished. Nineteen strains belonged to type 1, whereas only 5 strains belonged to type 2. The majority of strains isolated since 1991 in Belgium belong to type 1. No variations in fingerprinting patterns were observed within each group, confirming the highly conserved nature of the M. pneumoniae genome.
Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes
Authors: Lu J J , Bartlett M S , Shaw M M , Queener S F , Smith J W , Ortiz-Rivera M , Leibowitz M J , Lee C H
Published: January 2020

Small portions of the 18S and the 26S rRNA genes, the entire 5.8S rRNA gene, and internal transcribed spacers ITS1 and ITS2 (located between the 18S and 5.8S rRNA genes and between the 5.8S and 26S rRNA genes, respectively) of Pneumocystis carinii that infect humans were cloned and sequenced. The nucleotide sequences of the 18S, 5.8S, and 26S rRNA genes determined in the study were approximately 90% homologous to those of P. carinii that infect rats, while the sequences of ITS1 and ITS2 of P. carinii from the two different hosts were only 60% homologous. The 18S, 5.8S, and 26S rRNA gene sequences of P. carinii from 15 patient specimens were determined and were found to be identical to each other, whereas the ITS sequences were found to be variable. With the observed sequence variation, it was possible to classify the ITS1 sequences into two types and the ITS2 sequences into three types. P. carinii strains that had the same type of ITS1 sequence could have a different type of ITS2 sequence. On the basis of the sequence types of the two ITS regions, P. carinii from the 15 patients were classified into four groups. P. carinii from three patient specimens were found to contain two different ITS sequence patterns. More surprisingly, one additional specimen was found to have one ITS sequence typical of P. carinii isolates that infect humans and another typical of P. carinii isolates that infect rats. The studies indicate that it is possible to type P. carinii strains on the basis from one patient, suggesting that coinfection with more than one strain of P. carinii may occur in the same patient.
Molecular epidemiologic analysis of Vibrio cholerae O1 isolates by pulsed-field gel electrophoresis
Authors: Mahalingam S , Cheong Y M , Kan S , Yassin R M , Vadivelu J , Pang T
Published: January 2020

Isolates of Vibrio cholerae O1 El Tor from two well-defined cholera outbreaks in Malaysia were analyzed by using pulsed-field gel electrophoresis (PFGE). Isolates from sporadic cases occurring during the same time period were also studied. Digestion of chromosomal DNA from these isolates of V. cholerae O1 with restriction endonucleases NotI (5'-GCGGCCGC-3') and SfiI (5'-GGCCNNNN-3'), followed by PFGE, produced restriction endonuclease analysis (REA) patterns consisting of 13 to 24 bands (ranging in size from 46 to 398 kbp). Analysis of the REA patterns generated by PFGE after digestion with NotI and SfiI suggested the clonal nature and close genetic identity of the isolates obtained during each of the two outbreaks (Dice coefficient, 0.93 to 1.0). Although they had very similar REA patterns, the two outbreak clones were not identical. Isolates of V. cholerae O1 from sporadic cases, on the other hand, appeared to be much more heterogeneous (five different REA patterns detected in the five isolates tested; Dice coefficient, 0.31 to 0.81) than those obtained during the two outbreaks. We conclude that PFGE of V. cholerae O1 chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for molecular typing of V. cholerae isolates for epidemiological purposes.
Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing
Authors: Barrett T J , Lior H , Green J H , Khakhria R , Wells J G , Bell B P , Greene K D , Lewis J , Griffin P M
Published: January 2020

Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E. coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern. Twenty-five of 74 E. coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern. PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains. The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related. Phage typing and PFGE with additional enzymes were helpful in resolving this problem. While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates.
Detection of immunoglobulin M (IgM), IgA, and IgG Norwalk virus-specific antibodies by indirect enzyme-linked immunosorbent assay with baculovirus-expressed Norwalk virus capsid antigen in adult volunteers challenged with Norwalk virus
Authors: Gray J J , Cunliffe C , Ball J , Graham D Y , Desselberger U , Estes M K
Published: January 2020

Pre- and postexposure sera collected from 17 adult volunteers challenged with Norwalk virus as described previously (D. Y. Graham, X. Jiang, T. Tanaka, A. Opekun, P. Madore, and M. K. Estes, J. Infect. Dis. 170:34-43, 1994) were examined for Norwalk virus-specific immunoglobulin M (IgM), IgA, and IgG by indirect enzyme-linked immunosorbent assays with recombinant Norwalk virus antigen bound to the solid phase. Sixteen of the 17 volunteers had evidence of past infection, all presenting with preexisting IgG antibody of high avidity; only one volunteer had no evidence of previous infection. Virus infection was detected in 14 of the 16 volunteers with evidence of past infection, and 9 of the infected volunteers had symptomatic illness. A significant rise in both virus-specific IgA and IgG titers was detected after challenge in all of the volunteers who became ill. Five of the asymptomatic volunteers who were infected had rising titers of virus-specific IgG, but only two of the five had a concomitant rise in their virus-specific IgA antibody titers. Antibody rises were detectable in eight of nine ill volunteers 8 to 11 days after challenge but in the asymptomatic volunteers only after more than 15 days had elapsed. Virus-specific IgM was detected after challenge in all 14 infected volunteers. Between symptomatic and asymptomatic volunteers there were no significant differences in titers of virus-specific IgG and IgA in serum before challenge; however, there were significantly higher titers in symptomatic volunteers between 8 and > 90 days after challenge for virus-specific IgG and 8 and 24 days after challenge for virus-specific IgA.
Transportation of Helicobacter pylori cultures by optimal systems
Authors: Xia H X , Keane C T , Chen J , Zhang J , Walsh E J , Moran A P , Hua J S , Megraud F , O'Morain C A
Published: January 2020

Cultures of Helicobacter pylori on chocolate agar slants in bijou bottles and on chocolate agar plates inside BBL Campy Pouches were mailed from Dublin to Galway, Ireland; Bordeaux, France; and Beijing, China. Both systems maintained viability of H. pylori for at least 4 days under mailing conditions. Ninety percent of the isolates on the slants survived for 6 days, but only 30% of the isolates in the pouches survived. When the slants were stored at 4 degrees C after arrival, 50% of the isolates were recoverable 10 days after mailing. Failure of recovery was due to coccoid formation by the organisms. Contamination was not a problem in either system. Chocolate agar slants are considered the more suitable system for transporting H. pylori cultures, especially when transport time longer than 4 days is expected.
Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection
Authors: Mulder J , McKinney N , Christopherson C , Sninsky J , Greenfield L , Kwok S
Published: January 2020

A method for quantitating human immunodeficiency virus type 1 plasma viremia may be useful in monitoring disease progression and the responsiveness of patients to a therapeutic regimen or vaccine. A quantitative assay for viral RNA in plasma or sera that differs in several aspects from those reported previously was developed. First, whereas conventional reverse transcriptase-PCR assays involve a two-step process and use two enzymes, the method described uses a single enzyme, rTth DNA polymerase, for both reverse transcription and PCR. The reactions are carried out in a single tube and with a single buffer solution with uninterrupted thermal cycling. Second, uracil-N-glycosylase and dUTP are incorporated into the reaction mixtures to ensure that any carryover of DNA from previous amplifications will not compromise quantitation. Third, a quantitation standard is incorporated into each reaction mixture so that differences in amplification efficiency caused by sample interferents, variability in reaction conditions, or thermal cycling can be normalized. To ensure comparable amplification efficiency, the quantitation standard has the same primer-binding regions as the human immunodeficiency virus type 1 target and generates an amplified product of the same size and base composition. The probe-binding region was replaced with a sequence that can be detected separately. Fourth, a colorimetric detection format was modified to provide at least a four-log-unit dynamic range. The quantitative assay requires only a single amplification of the sample and can be completed in less than 8 h. The procedure was used on archival samples to demonstrate the viremic spike in acute infection and the suppressed levels of circulating virus following seroconversion.
Epidemiological study by pulsed-field gel electrophoresis of an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a geriatric hospital
Authors: Gouby A , Neuwirth C , Bourg G , Bouziges N , Carles-Nurit M J , Despaux E , Ramuz M
Published: January 2020

Twelve cases of infections caused by extended-spectrum beta-lactamase (ESBla)-producing Klebsiella pneumoniae were reported between August 1991 and March 1993 in the Geriatric Department of the Nimes University Hospital, where these bacterial had not been previously isolated. Restriction profiles of total genomic DNAs cleaved by XbaI and SpeI were compared by pulsed-field gel electrophoresis. The strains that were tested included the 12 isolates from K. pneumoniae-infected patients, strains recovered from rectal swabs of asymptomatic patients in the same ward, and strains isolated in other hospitals in Nîmes at the same time. The restriction profiles of the 12 isolates and those recovered from asymptomatic patients in the same ward were very similar. Over a period of more than 1 year, extended-spectrum beta-lactamases were not detected in K. pneumoniae isolates with restriction patterns different from that of the epidemic strain. It seems, therefore, that there was no transfer of a plasmid or a gene coding for ESBla to strains of K. pneumoniae that were different from the epidemic strain. At the same time, ESBla-producing K. pneumoniae isolates exhibiting restriction endonuclease profiles very different from that of the epidemic strain were isolated from other hospitals in Nîmes. None of these strains caused an outbreak. Pulsed-field gel electrophoresis, which allows precise characterization of strains beyond the species level, is a useful tool for studying the ESBla-producing K. pneumoniae strains involved in nosocomial outbreaks.
Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays
Authors: Khan A S , Moe C L , Glass R I , Monroe S S , Estes M K , Chapman L E , Jiang X , Humphrey C , Pon E , Iskander J K
Published: January 2020

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis.
Construction of polyepitope fusion antigens of human cytomegalovirus ppUL32: reactivity with human antibodies
Authors: Ripalti A , Ruan Q , Boccuni M C , Campanini F , Bergamini G , Landini M P
Published: January 2020

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides, can be valuable diagnostic material in the serology of HCMV infection (M. P. Landini, M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti, A. Necker, T. Lazzarotto, and B. Plachter, J. Clin. Microbiol. 28:1375-1379, 1990; M. P. Landini, T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa, J. Clin. Microbiol. 27:2324-2327, 1989; A. Ripalti, M. P. Landini, E. S. Mocarski, and M. La Placa, J. Gen. Virol. 70:1247-1251, 1989). In this work we addressed the question of whether the expression of more than one linear epitope on a single fusion protein could increase the reactivity of genetically engineered antigenic material with human antibody. To answer this question we fused sequences expressing two different epitopes contained in the basic phosphoprotein of 150 kDa encoded by UL32 (M. S. Chee, A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, T. Cerny, T. Hornsel, C. A. Hutchinson, T. Kouzarides, J. A. Martignetti, and B. G. Barrell, Curr. Top. Microbiol. Immunol. 154:125-169, 1990; G. Jahn, T. Kouzarides, M. Mach, B.-C. Scholl, B. Plachter, B. Traupe, E. Preddie, S. C. Satchwell, B. Fleckenstein, and B. G. Barrell, J. Virol. 61:1358-1367, 1987), ppUL32, which was repeatedly shown to be the strongest immunogen present in the viral particle. We also made fusions with sequences expressing a single epitope repeated once, twice, or three times. The different fusion proteins were tested with HCMV-positive human sera. We found that fusion proteins expressing different epitopes together were recognized by a larger number of serum specimens and with more intense reactions in Western blot (immunoblot) experiments. We also found evidence that expression on the same polypeptide of the two distinct epitopes produced a stronger antigen than the mere addition of two fusion proteins which each carried one copy of one of these epitopes. Furthermore, we found that while the same epitope expressed two or three times on the same fusion protein was not better recognized by immunoglobulin G than the single epitope, immunoglobulin M reactivities to the double and triple epitopes were enhanced.
Analysis of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by multilocus enzyme electrophoresis
Authors: Chooromoney K N , Hampson D J , Eamens G J , Turner M J
Published: January 2020

The genetic diversity of 74 Australian field isolates of Erysipelothrix rhusiopathiae and 22 reference strains for serovars of E. rhusiopathiae or Erysipelothrix tonsillarum was examined by multilocus enzyme electrophoresis. Four serovar reference strains of E. tonsillarum (strains KS 20 A, Wittling, Lengyel-P, and Bano 107 for serovars 25, 3, 10, and 22, respectively) were genetically distinct from E. rhusiopathiae. However, the E. tonsillarum reference strain for serovar 14 (Iszap-4) and the reference strain for serovar 13 (Pecs-56), which has been said to represent a new genomic species, were found to cluster with typical isolates and reference strains of E. rhusiopathiae. Our reference strain for serovar 7 (Rotzunge) was also genetically typical of E. rhusiopathiae, thus indicating that these serotype reactivities cannot be relied upon as a means of identifying isolates as E. tonsillarum. Australian field isolates of E. rhusiopathiae were genetically diverse. Those recovered from sheep or birds were more diverse than those isolated from pigs, and isolates of serovar 1 were more diverse than those of serovar 2. The diversity found among isolates of the same serovar and the presence of isolates of different serovars in the same electrophoretic types (ETs) indicated that serotyping of E. rhusiopathiae was unreliable for use as an epidemiological tool. Some ETs contained isolates recovered from different animal species. ET 41 contained 32.2% of the field isolates and two reference strains, indicating that this clone of E. rhusiopathiae is both widespread and commonly associated with disease in various species of animals.