bluuebunny/crossref_metadata_2025 huggingface dataset:
Adhesive properties and antibiotic resistance of Klebsiella, Enterobacter, and Serratia clinical isolates involved in nosocomial infections
Authors: Livrelli V , De Champs C , Di Martino P , Darfeuille-Michaud A , Forestier C , Joly B
Published: January 2020

Intestinal colonization by Klebsiella, Enterobacter, and Serratia (KES) strains is a crucial step in the development of nosocomial infections. We studied the adhesive properties, antibiotic resistance, and involvement in colonization or infection of 103 KES clinical isolates: 30 Klebsiella pneumoniae (29%), 16 Klebsiella oxytoca (15%), 30 Enterobacter aerogenes (29%), 14 Enterobacter cloacae (14%), and 13 Serratia sp. (13%) isolates. Half of them were resistant to several antimicrobial agents, including aminoglycosides and beta-lactam antibiotics. A total of 27 of 30 K. pneumoniae isolates (90%) adhered to the human cell line Intestine-407 (Int-407), while none of the K. oxytoca or E. aerogenes isolates and only 2 of the E. cloacae isolates adhered. Three adhesive patterns were observed for K. pneumoniae: an aggregative adhesion in 57% of the isolates, a diffuse adhesion in only one isolate, and a new pattern, localized adhesion, in 30% of the isolates. While most of the sensitive strains adhered with the aggregative phenotype, the localized pattern was associated with resistant K. pneumoniae isolates producing the CAZ-5 beta-lactamase. Furthermore, 45% of such localized-adhesion isolates were involved in severe infections. The distributions of type 1 and type 3 fimbriae, enteroaggregative E. coli, and cf29, pap, and afa/Dr adhesin-encoding genes were determined by using specific DNA probes. No relationship was found between the adhesive pattern and the production of specific fimbriae, suggesting that several unrecognized adhesive factors are involved. Our study indicates that special adhesive properties associated with resistance to antimicrobial agents could account for the pathogenicity of certain nosocomial strains.
Differentiation of Mycobacterium genavense and Mycobacterium simiae by automated mycolic acid analysis with high-performance liquid chromatography
Authors: Ritter D , Carlson L D , Logan B K , Ramos L S , Kilburn J O , Coyle M B
Published: January 2020

Mycobacterium genavense, a fastidious opportunist in patients with AIDS, cannot be identified by conventional biochemical methods. Computerized mycolic acid analysis by high-performance liquid chromatography offers an alternative that distinguishes the mycolic acid profile of M. genavense from those of all other organisms in the database developed at the Centers for Disease Control and Prevention.
Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis
Authors: Sreevatsan S , Escalante P , Pan X , Gillies D A , Siddiqui S , Khalaf C N , Kreiswirth B N , Bifani P , Adams L G , Ficht T , Perumaalla V S , Cave M D , van Embden J D , Musser J M
Published: January 2020

Automated sequence analysis of a 410-bp region of the axyR gene in 105 Mycobacterium tuberculosis complex isolates identified a polymorphic nucleotide that differentiated Mycobacterium bovis isolates from other complex members. All 29 M. bovis isolates sequenced had an adenine residue at nucleotide 285, whereas all 76 other complex isolates had a guanine residue. PCR-restriction fragment length polymorphism analysis of oxyR with restriction endonuclease AluI in an additional 255 complex isolates from widespread intercontinental sources confirmed and extended the unique association of adenine at position 285 with M. bovis isolates.
Dot-immunogold filtration assay as a screening test for syphilis
Authors: Huang Q , Lan X , Tong T , Wu X , Chen M , Feng X , Liu R , Tang Y , Zhu Z
Published: January 2020

A dot-immunogold filtration assay (DIGFA) for the rapid detection of reaginic antibody in the serum of syphilitic patients was developed. The assay was simple, rapid, and reproducible. The test completion time was 2 min, and the assay required no equipment. The positive dot was very obvious, and the results could easily be determined with the naked eye. A total of 350 serum samples were examined by DIGFA, the rapid plasma reagin test, and the fluorescent treponemal antibody-absorption test. The levels of agreement between DIGFA and the rapid reagin test and between DIGFA and the fluorescent treponemal antibody-absorption test were 100 and 98%, respectively. The results of clinical application indicated that DIGFA could be used as a routine screening test for syphilis.
Methicillin-resistant Staphylococcus aureus isolates recovered from a New York City hospital: analysis by molecular fingerprinting techniques
Authors: de Lencastre H , de Lencastre A , Tomasz A
Published: January 2020

Fifty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates collected at New York Hospital Medical Center of Queens in 1989 were analyzed by molecular fingerprinting techniques. Close to 70% of these isolates (38 of 55) shared a common pulsed-field gel electrophoretic pattern, carried the same mecA gene polymorph type II, were free of the transposon Tn554, and would not react with a mecI-specific gene probe. An additional five isolates shared all properties of the major MRSA clone except that they carried mecA gene polymorph type III. All these isolates had an extremely heterogeneous methicillin resistance phenotype that belonged to population analysis profile class 1 or 2. The rest of the 12 MRSA isolates showed a variety of chromosomal pulsed-field gel electrophoretic patterns that carried different mecA polymorphs and that also gave positive reactions with DNA probes for Tn554 and for the mecI gene. The molecular features of the majority MRSA clone suggest that it is an archaic MRSA isolate similar in features to early MRSA isolates recovered in the 1960s.
Clinical importance of increased sensitivity of BacT/Alert FAN aerobic and anaerobic blood culture bottles
Authors: McDonald L C , Fune J , Gaido L B , Weinstein M P , Reimer L G , Flynn T M , Wilson M L , Mirrett S , Reller L B
Published: January 2020

Two recent multicenter blood culture studies found that BacT/Alert FAN (FAN) bottles (Organon Teknika, Durham, N.C.) had increased yields in detecting bacteremia and fungemia compared with standard BacT/Alert (STD) bottles. Because the clinical importance of this increase in microbial recovery is unknown, we performed a retrospective analysis to determine the frequency with which FAN bottles were the sole means of detecting an episode of bacteremia. There were 1,047 positive blood cultures in which both study bottles were adequately filled and the organism isolated was judged to be the cause of sepsis: 240 (23%) were positive only in FAN bottles and 73 (7%) were positive only in STD bottles. Of a total of 664 episodes of bacteremia, 126 (19%) were identified only by FAN bottles and 43 (7%) were identified only by STD bottles (P < 0.0001). Episodes detected only by FAN bottles more often were recurrent events (23 of 126, or 18%) than episodes detected only by STD bottles (2 of 43, or 5%) (P < 0.05) and more commonly occurred in patients receiving theoretically effective antibiotic therapy (33 of 126 [26%] versus 4 of 43 [9%]) (P < 0.05). The medical records for patients with 127 of these episodes (92 FAN bottles only; 35 STD bottles only) were available for review. More than half of both FAN bottle-only (60 of 92, or 65%) and STD bottle-only (20 of 35, or 57%) episodes were judged to be clinically important. We conclude that FAN bottles improve the detection of bacteremia and that the majority of the additional episodes detected are clinically important. The benefits of the greater yield in specific patient populations must be balanced against the higher costs of FAN bottles.
Evaluation of an infectivity standard for real-time quality control of human immunodeficiency virus type 1 quantitative micrococulture assays. Participating Laboratories of The AIDS Clinical Trials Group
Authors: Scott W A , Brambilla D , Siwak E , Beatty C , Bremer J , Coombs R W , Farzadegan H , Fiscus S A , Hammer S M , Hollinger F B , Khan N , Rasheed S , Reichelderfer P S
Published: January 2020

Quantitative microculture assays of cryopreserved human immunodeficiency virus type 1-infected cell suspensions and culture supernatants were compared among seven assays sites. There was no significant change in titer during 1 year of storage. The overall standard deviation for infected cell suspensions was approximately 0.8 log10 virus titer. A method for detecting deviant assay results was developed and was used to identify two donor cell preparations (n = 54) that gave consistently low titers.
CDC group IIc: phenotypic characteristics, fatty acid composition, and isoprenoid quinone content
Authors: Hollis D G , Moss C W , Daneshvar M I , Wallace-Shewmaker P L
Published: January 2020

Twenty strains of glucose-utilizing, small gram-negative slightly pleomorphic rods that grew well aerobically and that were isolated from clinical specimens formed a phenotypically similar group that was designated CDC group IIc. The phenotypic characteristics of CDC group IIc were most similar to those of CDC groups IIe and IIh, the major differences being that CDC group IIc produced acid from sucrose, hydrolyzed esculin, and usually reduced nitrate. The CDC group IIc strains were analyzed by gas-liquid chromatography for their cellular fatty acid compositions, and all contained relatively large amounts of isobranched hydroxy and nonhydroxy acids. High-performance liquid chromatography and mass spectrometry analysis of the quinone extract showed menaquinone-6 as the major component. Both the cellular fatty acid and isoprenoid quinone compositions were consistent with the profiles of CDC groups IIe and IIh. Thirty percent of the isolates were from human blood.
Analysis of clinical and environment Yersinia isolates in the Republic of Georgia
Authors: Sulakvelidze A , Dalakishvili K , Barry E , Wauters G , Robins-Browne R , Imnadze P , Morris J G
Published: January 2020

The Center for Infectious Diseases Control, Georgian Ministry of Health, isolated 2,493 Yersinia enterocolitica and Y. enterocolitica-like strains, 22 Y. pestis strains, and 21 Y. pseudotuberculosis strains from 130,574 clinical and environmental samples. Analysis of 100 Y. enterocolitica and Y. enterocolitica-like strains showed none to be within traditional pathogenic biogroups or serogroups, and none carried genetic markers for virulence. However, some strains were enterotoxigenic in infant mice, while others were associated with prolonged carriage in adult mice.
Detection of Legionella pneumophila serogroup 1 antigen in nonconcentrated urine and urine concentrated by selective ultrafiltration
Authors: Domínguez J A , Manterola J M , Blavia R , Sopena N , Belda F J , Padilla E , Giménez M , Sabrià M , Morera J , Ausina V
Published: January 2020

We evaluated a commercially available radioimmunoassay technique for detecting the soluble antigen of Legionella pneumophila serogroup 1 using concentrated and nonconcentrated urine. Concentration was achieved by selective ultrafiltration. The sensitivity of the technique was 60.9% for nonconcentrated urine and 80.4% for concentrated samples, with 100% specificity in both cases.
Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR
Authors: Yoo H S , Lee S U , Park K Y , Park Y H
Published: January 2020

Clostridium perfringens has been classified into five toxigenic types (A through E) on the basis of its capability to produce major lethal toxins (alpha, beta, epsilon, and iota toxins). Seroneutralization with mice or guinea pigs has been used to type each toxin, but this conventional method has some disadvantages. Therefore, we used a molecular biological technique to type the bacterium in the present study. A multiplex PCR was developed for this purpose. This method has several advantages in comparison with seroneutralization with mice or guinea pigs. By this method, we also investigated the most prevalent type(s) of the organism in Korean calves, piglets, and chickens showing clinical symptoms such as diarrhea, enterotoxemia, and necrotic enteritis. Only type A was isolated from calves and chickens, while type C (2 of 14 isolates), in addition to type A, was isolated from piglets. These results suggested that seroneutralization could be replaced by our new method and that type A of C. perfringens is the most prevalent type in livestock in Korea.
Comparison of in-house and commercial sample preparation and PCR amplification systems for detection of human immunodeficiency virus type 1 DNA in blood samples from Tanzanian adults
Authors: Lyamuya E , Bredberg-Rådén U , Albert J , Grankvist O , Msangi V , Kagoma C , Mhalu F , Biberfeld G
Published: January 2020

This study compared the performance of several in-house nested PCR systems and the Amplicor human immunodeficiency virus type 1 (HIV-1) PCR kit in the detection of HIV-1 DNA in Tanzanian samples prepared by two different methods. All six of the in-house primer sets evaluated had a higher sensitivity for HIV DNA detection in samples prepared by the Amplicor PCR sample preparation method than in those prepared by the Ficoll-Isopaque (FIP) density gradient centrifugation method. A sensitivity of 100% was achieved by combining two in-house primer sets. The sensitivity of the standard Amplicor HIV-1 PCR kit was only 59%, whereas a modified Amplicor HIV-1 PCR test had a sensitivity of 98%. Our data show that Tanzanian samples prepared by the Amplicor preparation method are more suitable for HIV-1 PCR testing than samples prepared by the FIP method. The modified, but not the standard, Amplicor HIV-1 PCR kit provides an alternative to the nested in-house PCR technique for the diagnosis of HIV infection.
Rabbit model for Chlamydia pneumoniae infection
Authors: Fong I W , Chiu B , Viira E , Fong M W , Jang D , Mahony J
Published: January 2020

A rabbit model was established for Chlamydia pneumoniae infection that may be helpful to understand the pathogenesis of disease in humans. Twelve, pathogen-free, 1-month-old New Zealand White rabbits were inoculated with 1.0 x 10(7) to 5.0 x 10(7) CFU of purified C. pneumoniae (ATCC strain VR 1310) via the nasopharynx (1 rabbit died immediately postinoculation, and 11 were available for study). Five controls were inoculated with the carrier buffer. Ten of the 11 study rabbits demonstrated serological evidence of acute infection (immunoglobulin G antibodies, 1:8 to > 1:16), with the weakest response at 7 days and the strongest response at 28 days, whereas none of the controls showed any seroconversion. Study animals were sacrificed in batches of three, on days 7, 14, 21, and 28, but controls were sacrificed on days 7 and 28. Two-thirds of the animals demonstrated evidence of bronchiolitis and pneumonia on days 7 and 14 and resolution by day 21. Two study rabbits demonstrated, on histology, early and intermediate lesions of atherosclerosis: one animal (day 7) showed the accumulation of foamy macrophages (fatty streak) in the arch of the aorta, and the other animal (day 14) showed spindle cell proliferation of smooth muscle cells (intermediate lesion). Focal periaortitis was seen in the same animal (day 7). C. pneumoniae elementary bodies were demonstrated by immunocytochemical stain in the lungs (n = 2), liver (n = 3), spleen (n = 5), and aorta (n = 2), one of which corresponded to the intermediate lesion. C. pneumoniae was cultured from the lungs (n = 2), liver (n = 2), spleen (n = 2), and aortic arch (n = 1). All histopathological, immunocytochemical, and cultural studies were negative in the controls. Hence, the rabbit provides a useful animal model for the study of C. pneumoniae infection and its complications, particularly atherosclerosis.
Controlled clinical laboratory comparison of BACTEC plus aerobic/F resin medium with BacT/Alert aerobic FAN medium for detection of bacteremia and fungemia
Authors: Jorgensen J H , Mirrett S , McDonald L C , Murray P R , Weinstein M P , Fune J , Trippy C W , Masterson M , Reller L B
Published: January 2020

Blood specimens collected from adult patients with suspected sepsis in four medical centers were inoculated into BACTEC Plus/F and BacT/Alert FAN aerobic culture bottles. Both bottles of 7,401 bottle pairs contained the prescribed blood volume of 8 to 12 ml. Bottles were incubated in their respective instruments for a standard 7-day protocol or until the instruments signaled that they were positive. A total of 720 isolates that were judged to represent true infections were recovered from 338 patients; 451 isolates were recovered from both bottles, 143 were recovered from only the Plus/F bottle, and 126 were recovered from only the FAN bottle (P was not significant). Although more Histoplasma capsulatum isolates were recovered from Plus/F bottles (P < 0.005), there were no other statistically significant differences in recovery rates of individual species or groups of organisms between the two systems. Of 329 monomicrobic patient septic episodes, 244 episodes were detected by both blood culture systems, 40 were detected only by the BACTEC system, and 45 were detected only by the BacT/Alert system (P was not significant). There was no significant difference between the two systems in the detection of septic episodes among patients receiving antibiotic therapy at the time of blood cultures. Of the cultures found to be positive within the first 72 h of incubation, detection was on average earlier by the BACTEC system (16.9 h) than by the BacT/Alert system (18.7 h). Larger differences in average time to detection were seen with streptococci (10.7 h by the BACTEC system and 17.9 h by the BacT/Alert system) and yeasts (an average of 29.4 h by the BacT/Alert system versus 37.2 h by the BACTEC system). With the exception of the differences noted above, BACTEC Plus/F aerobic resin and BacT/Alert aerobic FAN blood culture bottles were comparable in their abilities to recover aerobic and facultative organisms.
Occurrence and detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find
Authors: Coudron P E , Moland E S , Sanders C C
Published: January 2020

A total of 907 consecutive isolates of members of the family Enterobacteriaceae recovered during a 20-week period were tested for production of extended-spectrum beta-lactamases (ESBLs) by the double-disk (DD) potentiation method. Of 84 DD-positive isolates, 83 (9.2%) produced ESBLs based on isoelectric focusing. SHV-derived ESBLs and several TEM-derived ESBLs were present in nine species, including the first isolate of Citrobacter koserii and Morganella morganii known to harbor an SHV-derived ESBL. Results of testing 58 nonrepeat isolates for ESBL production by several recommended methods were as follows (percent detected in parentheses): DD method with aztreonam (95), ceftazidime (79), ceftriaxone (88), or cefpodoxime (90); broth microdilution method with ceftazidime (86) or cefotaxime (91) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (78), ceftazidime (79), ceftriaxone (83), or cefpodoxime (98) or a novel concentration (5 microg) of ceftazidime (88). In three instances during an extended part of the study, an ESBL-producing isolate and a non-ESBL-producing isolate of the same species were recovered from a single blood culture bottle. These data indicate that ESBLs occur in several species of Enterobacteriaceae and at a relatively high incidence at our institution and that the standard disk diffusion method with cefpodoxime and the DD method with several beta-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of Enterobacteriaceae.
Comparison of major antigenic proteins of six strains of the human granulocytic ehrlichiosis agent by Western immunoblot analysis
Authors: Zhi N , Rikihisa Y , Kim H Y , Wormser G P , Horowitz H W
Published: January 2020

The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE patterns with respect to 49- and 47-kDa proteins. No. 3 and USG isolates lacked the 47-kDa protein, and no. 6 isolate lacked the 49-kDa protein. Both 49- and 47-kDa bands were absent in no. 2 isolate. Western blot results with seven different sera, including five convalescent-phase sera from these patients, one dog anti-USG isolate, and one horse anti-BDS isolate, showed that all major antigens in six isolates were recognized by all sera. However, the molecular sizes and the numbers of major antigens recognized varied among the six isolates. Overall, HGE agent no. 3, 6, 11, and 13, and USG isolates had similar patterns, with 1 or 2 major antigens with molecular masses of around 49 and 47 kDa. No. 2 isolate was quite distinct in having a major antigen of 43 kDa. This indicates that although these antigenic epitopes are all cross-reactive among strains, the HGE agent has a strain pleomorphism in its major antigenic proteins. The major antigen profiles of the outer membrane protein fractions and of whole organisms of six HGE agent isolates were similar, suggesting that 49- and 47-kDa major antigens are the outer membrane proteins of the HGE agent.
Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays
Authors: Zuerner R L , Bolin C A
Published: January 2020

Genetic variability among Leptospira interrogans (sensu stricto) serovars was assessed by Southern blot hybridization and PCR analyses. The experiments used probes directed to sequences in a recently described insertion element, IS1500. Hybridization analysis showed that IS1500 was present on polymorphic fragments and that differences in these patterns could be used to identify serovars. Hybridization analysis was also useful in discriminating between serovar pomona type kennewicki isolates, making possible the identification of 15 previously unrecognized genetic groups. A PCR assay was developed in which the primers are positioned near the terminal inverted repeats of the element and directed outward. This assay yielded characteristic amplification patterns from isolates, allowing them to be identified. We applied these assays to several new animal isolates of L. interrogans from Nicaragua, which recently had an outbreak of human leptospirosis. Three groups of isolates were identified: one strain of serovar pomona type kennewicki and two genetically distinct groups of isolates which may be genetic intermediates between serovars canicola and portlandvere. The IS-based typing assays described should be useful for epidemiological analysis of leptospirosis.
Detection of lipoarabinomannan antibodies in patients with newly acquired tuberculosis and patients with relapse tuberculosis
Authors: Julián E , Matas L , Ausina V , Luquin M
Published: January 2020

A commercially available dot immunoassay that employs the lipoarabinomannan antigen was evaluated for the serologic diagnosis of tuberculosis. The test showed a high specificity (100%); however, its sensitivity was low (18.5%). Antibodies to lipoarabinomannan were detected in the sera of 7 of 71 patients with newly acquired tuberculosis and in sera of 10 of 21 patients with relapse tuberculosis. It has been shown by others that sera from patients with relapse tuberculosis had a higher concentration of antibodies and reacted with a greater variety of antigens (native culture filtrates of Mycobacterium tuberculosis H37Rv) than did sera from patients with newly acquired tuberculosis. Our data confirm the results of these previous studies as far as lipoarabinomannan is concerned. We conclude that the differences in the production of antibodies shown by the two groups of tuberculous patients (new and relapse) must be taken into account when assessing the usefulness of serologic tests for the diagnosis of tuberculosis.
Natural transmission of Pneumocystis carinii in nonimmunosuppressed animals: early contagiousness of experimentally infected rabbits (Oryctolagus cuniculus)
Authors: Ceré N , Polack B , Chanteloup N K , Coudert P
Published: January 2020

Airborne transmission of Pneumocystis carinii has been established, but the infective form and the sources of infection remain unknown. Animal models for studies of P. carinii have previously been limited to immunosuppressed rodents; however, this study was performed with nonimmunodepressed P. carinii-free rabbits. This study was aimed at determining (i) the delay between inoculation of animals (day zero [D0]) and the onset of contagiousness and (ii) the end of contagiousness of these animals (donors). Five-week-old rabbits were used as contact animals and were housed with the donors. The cohabitation periods were for 4 or 5 days from D0 to D4, D4 to D8, D8 to D13, D13 to D18, and D18 to D22. The highest parasite burdens were observed in contact animals housed with donors from D8 to D13 or D13 to D18. This period (8th to 18th day following the day of inoculation of donors) might correspond to the highest phase of contagiousness of donors.
Citrobacter sedlakii meningitis and brain abscess in a premature infant
Authors: Dyer J , Hayani K C , Janda W M , Schreckenberger P C
Published: January 2020

Citrobacter sedlakii was isolated from blood and cerebrospinal fluid cultures of a 5-day-old premature infant with sepsis, meningitis, and brain abscess. This newly described organism was difficult to identify due to discrepancies between the Vitek and API 20E identification systems. To our knowledge, this is the first report of the isolation of C. sedlakii from cerebrospinal fluid.
Evaluation of commercially available and in-house reverse transcription-PCR assays for detection of hepatitis G virus or GB virus C
Authors: Forns X , Tan D , Alter H J , Purcell R H , Bukh J
Published: January 2020

Serum samples from 96 Spanish hemodialysis patients, as well as serial dilutions of RNA extracted from a reference strain of hepatitis G virus (HGV), were tested for HGV or GB virus C (GBV-C) RNA. Two different reverse transcription (RT)-PCR-based methods of detection were compared for the ability to detect RNA extracted from the samples: an RT-nested PCR assay with primers derived from the 5' noncoding region (5'NC) or nonstructural region 3 (NS3) sequences and a commercially available RT-PCR assay with primers derived from the 5'NC or NS5A sequences. When RT-nested PCR was performed on 10-fold serial dilutions of RNA from the HGV reference strain, the last positive dilution was 10(-7) to 10(-8). With the commercial RT-PCR assay, the last positive dilution was 10(-6) to 10(-7). When equal amounts of RNA extracted from serum samples from 96 hemodialysis patients were tested for HGV or GBV-C RNA, 25 patients (26%) were positive by the RT-nested PCR. However, only 21 (84%) of these 25 positive patients were positive for HGV or GBV-C by the commercial RT-PCR assay. Analysis of the 5'NC and NS3 sequences amplified by RT-nested PCR demonstrated that all but two positive patients had unique HGV or GBV-C sequences. In summary, RT-nested PCR and a commercially available RT-PCR assay for HGV or GBV-C gave concordant results for 96% of the patients tested.
Colonization of skin by Helcococcus kunzii
Authors: Haas J , Jernick S L , Scardina R J , Teruya J , Caliendo A M , Ruoff K L
Published: January 2020

In order to investigate the role of Helcococcus kunzii as a colonizer of skin and as a possible participant in diabetic foot ulcers, we used a selective medium to culture both lower- and upper-extremity skin from a study group of podiatry patients (60 diabetics and 60 nondiabetics) and a control group of 50 healthy volunteers. Although differences in colonization were not statistically significant, a trend toward higher colonization rates in the group of podiatry patients was noted. H. kunzii appears to preferentially colonize the skin of the feet, and while its pathogenic role in diabetic foot ulcers is difficult to establish, it may be a previously unrecognized component of the polymicrobial flora characteristically isolated from patients with these infections.
Inhibitory effects of polyoxyethylene stearate, PANTA, and neutral pH on growth of Mycobacterium genavense in BACTEC primary cultures
Authors: Realini L , Van Der Stuyft P , De Ridder K , Hirschel B , Portaels F
Published: January 2020

We report on the influences of polyoxyethylene stearate (POES), PANTA, and pH on primary cultures of Mycobacterium genavense in BACTEC vials. As a model for primary cultures from tissue, seven different strains first isolated from AIDS patients (five from Switzerland and two from the United States) were inoculated into nude mice in order to obtain large amounts of bacilli to test different conditions simultaneously. Our results demonstrate that the size of the inoculum (10[6] acid-fast bacilli/vial), an acid pH (pH 6.0), and the absence of additives (POES and PANTA) significantly (P < 0.001) increased the probability of a successful culture in 1 month, considering growth index (GI) of > or =100 or a GI of > or =999 as criterion of success. In logistic regression analysis, all factors maintained a significant (P < 0.001) independent effect, and no interactions were observed between them. The best conditions for the primary cultures of M. genavense were the use of Middlebrook 7H12 medium at pH 6.0 without any additives.
Report of spores of Henneguya salminicola (Myxozoa) in human stool specimens: possible source of confusion with human spermatozoa
Authors: McClelland R S , Murphy D M , Cone D K
Published: January 2020

The spores of Henneguya salminicola, a common tissue parasite of salmonid fishes in the northern hemisphere, were observed in stool specimens from two different patients with diarrhea. The spores' superficial resemblance to human spermatozoa resulted, in one instance, in an incorrect report, leading to suspicion of sexual abuse. H. salminicola spores and human spermatozoa can be differentiated on the basis of size, morphology, and staining characteristics. Laboratory personnel who perform microscopic examinations of stool specimens for ova and parasites should be aware that spores of H. salminicola may be seen from time to time.
Performance of the Amplicor human immunodeficiency virus type 1 PCR and analysis of specimens with false-negative results
Authors: Barlow K L , Tosswill J H , Parry J V , Clewley J P
Published: January 2020

Over a 4-year period, the Roche Amplicor kit was used in a United Kingdom reference laboratory for the detection or confirmation of human immunodeficiency virus (HIV) type 1 infection, particularly in infants born to HIV-infected mothers. Of 408 specimens from adults and older children tested, the 122 seronegative specimens were all Amplicor negative. Of the 286 seropositive specimens, 268 were Amplicor positive. On the basis of these results, the Amplicor assay has a specificity of 100% and a sensitivity of 93.7%. In addition, for 247 specimens from infants and young children, serological results may not have been diagnostic because of placental transfer of maternal antibodies. Forty-eight were Amplicor positive, and of the 199 Amplicor-negative specimens, 19 were assumed to be false negative on the basis of clinical data, serological markers (including p24 antigen), and/or results for previous or follow-up specimens. This represents a sensitivity of 75% for the Amplicor test for specimens from patients under 2 years of age. Of these 37 false-negative specimens plus 2 specimens from other laboratories, 31 could be characterized by amplifying extracted material from them by an in-house nested gag PCR spanning the Amplicor target region. The amplicons were sequenced and found to represent subtypes A (35.5%), B (22.6%), C (22.6%), D (16.1%), and G (3.2%). False-negative results by the Amplicor assay may be ascribed to low-target copy number, the physical behavior of one primer (SK462), and sequence variation in the target region of the other primer (SK431).
Use of pulsed-field gel electrophoresis to determine genomic diversity in strains of Helicobacter hepaticus from geographically distant locations
Authors: Saunders K E , McGovern K J , Fox J G
Published: January 2020

In 1992 a helical microorganism associated with chronic active hepatitis and a high incidence of hepatocellular tumors was identified in the hepatic parenchyma of A/JCr mice. By using biochemical tests, phenotypic characterization, and 16S rRNA gene sequence analysis, the organism was classified as a novel Helicobacter species and named Helicobacter hepaticus. Recent surveys completed in our laboratory indicate that H. hepaticus is widespread in academic and commercial mouse colonies. The aim of this study was to examine the H. hepaticus genome by pulsed-field gel electrophoresis (PFGE) to determine the degree of genomic variation and genomic size. This technique has been used to identify significant genomic diversity among strains of Helicobacter pylori and to demonstrate only slight genomic diversity among strains of Helicobacter mustelae. Genomic DNAs from 11 isolates of H. hepaticus from the United States, Germany, France, and The Netherlands were subjected to PFGE after digestion with SmaI. Isolates from three independent sources within the United States had very similar PFGE patterns, suggesting that the genomic DNAs of these isolates are conserved. Genomic DNA isolated from a fourth source within the United States had a PFGE pattern different from those of the other U.S. isolates. Isolates obtained from Germany, France, and The Netherlands had PFGE patterns that differed markedly from those of the U.S. isolates and from one another. The use of DNA fingerprinting may be useful in subsequent epidemiological studies of H. hepaticus when the source and method of spread of this murine pathogen need to be ascertained. By PFGE, the genomic size of H. hepaticus is estimated to be roughly 1.3 Mb, which compares to 1.67 Mb for H. pylori and 1.7 Mb for H. mustelae.
Frequency and natural history of rhinovirus infections in adults during autumn
Authors: Arruda E , Pitkäranta A , Witek T J , Doyle C A , Hayden F G
Published: January 2020

Human rhinovirus (HRV) accounts for a significant portion of common-cold illness, with the peak incidence being in the early fall. Three hundred forty-six adults who had self-diagnosed colds of 48 h or less were enrolled in a study during September and October 1994 to determine the frequency and clinical course of HRV infections. Nasal wash specimens for viral culture and reverse transcription-PCR (RT-PCR) for HRV RNA and human coronavirus OC43 and 229E RNA detection were collected on enrollment, and participants recorded their symptoms twice daily for 14 days. Middle ear pressure (MEP) was measured with a digital tympanometer on days 1 and 7. Picornaviruses (224 HRV and 7 enterovirus isolates) were detected by culture in 67% (231 of 346) of the subjects. Among 114 samples negative by culture, HRV was detected by RT-PCR in 52 (46%) for an overall picornavirus infection rate of 82% (283 of 346 subjects). Among the remaining 62 negative samples, human coronavirus RNA was detected by RT-PCR in 5 patients, so that 288 (83%) of patients had documented viral infection. The first symptom noticed most often was sore throat (40%) in HRV culture- or PCR-positive patients and stuffy nose in HRV-negative patients (27%). No differences in symptom scores over time or in the presence of individual symptoms were noted between groups. The median duration of the cold episodes was 11 days in HRV culture-positive patients, 9.5 days in HRV RT-PCR-positive patients, and 11.5 days in HRV-negative patients. On enrollment, abnormal MEPs (< or = -100 or > or = +100 mm of H2O) were found for 21% of HRV culture-positive patients, 14% of HRV RT-PCR-positive patients, and 10% of HRV-negative patients. No important differences in the clinical course of HRV culture-positive, HRV culture-negative and RT-PCR-positive, or HRV-negative colds were found. These results represent the highest frequency of virologically confirmed natural colds to date and document the importance of rhinoviruses as the cause of colds during fall months.
Molecular evidence and clinical significance of herpesvirus coinfection in the central nervous system
Authors: Tang Y W , Espy M J , Persing D H , Smith T F
Published: January 2020

A total of 60 cerebrospinal fluid (CSF) specimens from patients manifesting symptoms resembling viral central nervous system (CNS) disease were examined for the presence of herpes simplex virus (HSV), human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus, varicella-zoster virus, Borrelia burgdorferi, and Tropheryma whippelii DNA by PCR. Of 30 specimens which were selected on the basis of HSV DNA positivity, 2 were concomitantly positive for HHV-6 DNA and 1 was positive for EBV DNA. In the three specimens positive for more than one herpesvirus, amplicons generated with virus-specific primer sets hybridized specifically to the corresponding virus-specific probe. Sequence analysis of the two amplified DNA fragments demonstrated that they were derived from distinct herpesviruses. Of 22 patients with clinically diagnosed encephalitis, 2 of 3 patients coinfected with HSV and HHV-6 died, compared to 1 of 19 (5%) patients infected with only HSV. Of 30 CSF specimens that were negative for HSV DNA, EBV DNA was detected in one sample. These data indicated the presence of DNA specific for two distinct herpesviruses in the same CSF specimen, providing molecular evidence that coinfection with this group of viruses may occur in the CNS.
Laboratory diagnosis of central nervous system infections with herpes simplex virus by PCR performed with cerebrospinal fluid specimens
Authors: Mitchell P S , Espy M J , Smith T F , Toal D R , Rys P N , Berbari E F , Osmon D R , Persing D H
Published: January 2020

Until recently, the laboratory diagnosis of central nervous system (CNS) infections with herpes simplex virus (HSV) has been limited by poor sensitivity and/or specificity. We assessed the diagnostic utility of PCR for detection of HSV in over 2,100 specimens referred to the Mayo Clinic from August 1993 to May 1996. DNA extracted from cerebrospinal fluid (CSF) samples with IsoQuick was amplified by PCR with oligonucleotide primers directed to the DNA polymerase gene of HSV, yielding a 290-bp amplicon. HSV DNA was detected in 150 (135 by gel electrophoresis, 15 by Southern blotting only) of 2,106 (7.1%) specimens. PCR-positive CNS disease occurred in patients ranging in age from 13 days to 89 years; 59% of the cases occurred in patients between the ages of 30 and 69, and 21 (14%) of the patients were infants. Genotype analysis was not routinely performed; however, amplification of a 335-bp product within the thymidine kinase gene of HSV revealed 13 positions within a span of 80 nucleotides that accurately identified the two serotypes of the virus according to 14 reference strains. We conclude that PCR detection of HSV DNA in CSF specimens should be considered an emerging "gold standard" for the laboratory diagnosis of CNS infections with this virus.
Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens
Authors: Monteiro L , Cabrita J , Mégraud F
Published: January 2020

PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. The three assays were based on the amplification of a fragment of the ureC gene of H. pylori and a colorimetric hybridization assay. The first assay (GEN-ETI-K DNA enzyme immunoassay; Sorin, Sallugia, Italy) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody. The second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection. Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA labelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate. The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fold dilutions of DNA from H. pylori CIP 101260, and the specificity was assessed with different urease-positive bacteria. Biopsy specimens from 199 patients were tested; 106 were classified as H. pylori positive, and 93 were classified as H. pylori negative by culture and/or histological examination as the "gold standard." The receiving operating characteristic curve was used to determine the best cutoff point for each assay. The detection of PCR products by colorimetric hybridization increases the sensitivity up to 100-fold compared to that with gel electrophoresis. The results are rapid (4 h) and easy to interpret and can be automated.
Detection and identification of Candida species in experimentally infected tissue and human blood by rRNA-specific fluorescent in situ hybridization
Authors: Lischewski A , Kretschmar M , Hof H , Amann R , Hacker J , Morschhäuser J
Published: January 2020

Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in a mouse infected with the closely related species C. parapsilosis. Conversely, the C. parapsilosis-specific probe detected fungal cells in the deep organs of a mouse infected with C. parapsilosis but not in the deep organs of a C. albicans-infected mouse. In addition, the C. albicans-specific probe was used to detect this species in human blood spiked with yeast cells by a lysis-filtration assay and subsequent fluorescent in situ hybridization. By this assay, as few as three yeast cells per 0.5 ml of blood were consistently detected. Our results demonstrate that fluorescent in situ hybridization with species-specific rRNA-targeted oligonucleotide probes provides a novel, culture-independent method for the sensitive detection and identification of Candida species in clinically relevant material.
Serotyping of group A rotaviruses in Egyptian neonates and infants less than 1 year old with acute diarrhea
Authors: Radwan S F , Gabr M K , El-Maraghi S , El-Saifi A F
Published: January 2020

Group A human rotavirus G serotypes were detected in stool specimens from neonates and infants with and without acute diarrhea in Cairo by using monoclonal antibodies in an enzyme-linked immunosorbent assay. Serotypes G1 and G4 predominated in all age groups. Mixed (G1 plus G4) and nontypeable specimens represented 16.1 and 38.7% of the total number serotyped, respectively.
Immunoblot analysis of Leishmania panamensis antigens in sera of patients with American cutaneous leishmaniasis
Authors: Isaza D M , Restrepo M , Mosca W
Published: January 2020

Sera from 86 Colombian patients with parasitologically confirmed cutaneous leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania panamensis was used as the antigen. Sera from patients with Chagas' disease and sera from patients with no record of infection with trypanosomatids were also studied. The sera from patients with cutaneous leishmaniasis specifically recognized fractions of 120 kDa (76.7%), 123 and 129 kDa (69.7%), 138 kDa (61.6%), 141 kDa (53.4%), and 78 kDa (44.1%). No band common to all patients infected with Leishmania parasites was found at the time of diagnosis. Likewise, the pattern of immunoblot change after the patients were treated and apparently cured with meglumine antimoniate (Glucantime) was studied by evaluating sera pretreatment and 1 year posttreatment. Only minor changes in the color intensity at the same serum dilution between pre- and posttreatment sera were found, although the antibody titers by indirect immunofluorescence were negative for the posttreatment sample. This study shows that Western blot analysis is a more sensitive test for the detection of anti-Leishmania antibodies. However, the importance of whether the presence of antibodies correlates with the presence of Leishmania antigens could not be resolved by the data obtained from this study.
Distinctive IS200 insertion between gyrA and rcsC genes in Salmonella typhi
Authors: Calva E , Ordoñez L G , Fernandez-Mora M , Santana F J , Bobadilla M , Puente J L
Published: January 2020

While probing the vicinity of ompC, a copy of the IS200 insertion element was found between the gyrA and rcsC genes of Salmonella typhi, the causal agent of typhoid fever. This distinctive feature was conserved throughout 63 S. typhi isolates of different geographical origins and was absent from 46 other Salmonella serotypes, including those most associated with human infections, as well as from 19 other enteric bacteria. Furthermore, the location of this IS200 copy corresponds to a constant band, present throughout the 14 PstI S. typhi IS200 fingerprints, encompassing several Vi phage types. Interestingly, an apparently unrelated serotype not frequently associated with human disease, Salmonella weltevreden, contained an IS200 copy at the same position, although it was accompanied by an additional segment of cryptic DNA. On the basis of these findings, PCR assays were designed for molecular typing of S. typhi, and these are potentially useful in studying the epidemiology of typhoid fever.