bluuebunny/crossref_metadata_2025 huggingface dataset:
Human cytomegalovirus (CMV) DNA in plasma reflects quantity of CMV DNA present in leukocytes
Authors: Zipeto D , Morris S , Hong C , Dowling A , Wolitz R , Merigan T C , Rasmussen L
Published: January 2020

A quantitative DNA amplification assay for human cytomegalovirus (CMV) DNA has been used to evaluate the relationship between quantities of CMV DNA in plasma and those in infected leukocytes (WBC) from human immunodeficiency virus-infected patients. The target sequence for DNA amplification was a region of the immediate-early 1 gene of CMV. The quantitation assay uses an internal control that is coamplified with each patient sample DNA and contains a sequence for detection by colorimetric hybridization with the same bases, but in different order than in the CMV immediate-early 1 region used for hybridization of amplified patient sample DNA. Results showed that patients with CMV disease had more CMV DNA in both WBC and plasma than those without disease. However, in this study, copy numbers of CMV DNA in WBC were higher than those in plasma. The gB and gH variants were the same in plasma and WBC.
Partial nucleotide sequencing of six subtype 2c hepatitis C viruses detected in Italy
Authors: Cammarota G , Maggi F , Vatteroni M L , Da Prato L , Barsanti L , Bendinelli M , Pistello M
Published: January 2020

The great majority of 121 hepatitis C virus (HCV) isolates obtained from 117 Italian patients with community-acquired infection could readily be typed by genotype-specific PCR. Subtype 1b was dominant (74 isolates); subtypes 2b, 2a, and 1a followed, with 19, 14, and 8 isolates, respectively. The six isolates that remained untyped by this method were classified as subtype 2c on the basis of sequence analysis of PCR amplicors obtained from the core and NS5 genes. These findings indicate that HCV subtype 2c has a relatively high prevalence in Italy. Sequencing the core region from positions 160 to 259 is sufficient to distinguish subtype 2c from other known HCV genotypes.
Comparison of six different PCR methods for detection of Pneumocystis carinii
Authors: Lu J J , Chen C H , Bartlett M S , Smith J W , Lee C H
Published: January 2020

We have recently developed a nested PCR method which amplifies internal transcribed spacers (ITS) of the ribosomal RNA genes of Pneumocystis carinii. To determine whether this PCR method can be used to diagnose P. carinii infections, we examined 30 bronchoalveolar lavage (BAL) specimens that were shown microscopically to contain P. carinii organisms by the P. carinii ITS PCR (Pc-ITS-PCR) and five other PCR methods that have been described for detecting P. carinii in clinical specimens. The targets of these PCR methods are portions of 18S rRNA, mitochondrial (mt) rRNA, 5S rRNA, thymidylate synthase (TS), and dihydrofolate reductase (DHFR). We also examined five different fungi, including Saccharomyces cerevisiae, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus to determine the specificity of these six PCR methods for P. carinii. All 30 BAL specimens were positive by both the Pc-ITS-PCR and the 18S rRNA gene PCR, whereas only 26 (87%), 18 (60%), 10 (33%), and 7 (23%) of 30 BAL specimens were positive by mt rRNA gene PCR, TS gene PCR, 5S rRNA gene PCR, and DHFR gene PCR, respectively. Although the 18S rRNA gene PCR had the same sensitivity as the Pc-ITS-PCR, it nonspecifically amplified S. cerevisiae and C. albicans. The TS gene PCR also produced false-positive PCR results with C. albicans and C. neoformans. None of the other PCR methods (Pc-ITS-PCR, mt rRNA gene, 5S rRNA gene, and DHFR gene PCR) amplified the control fungal DNA. Considering both sensitivity and specificity, we conclude that Pc-ITS-PCR is the most effective of the six PCR methods evaluated in this study for the detection of P. carinii in BAL specimens.
Evaluation of Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay and comparison with cell culture and Syva Chlamydia MicroTrak II EIA in high- and low-risk populations
Authors: Chan E L , Brandt K , Horsman G
Published: January 2020

Seven hundred thirty-two female urogenital samples were collected for Chlamydia trachomatis testing by both the Sanofi Diagnostics Pasteur (Chaska, Minn.) Chlamydia Microplate EIA by the shortened protocol and the Syva (San Jose, Calif.) MicroTrak II EIA, and the results were compared with those obtained by cell culture. For the analysis of samples from female patients, the patients were divided into high- and low-risk categories. An additional 121 male urethral samples were collected and tested by the Sanofi Microplate EIA and cell culture; for the analysis of samples from male patients, the patients were divided into asymptomatic and symptomatic categories. All specimens positive by enzyme immunoassay (EIA) were confirmed by a blocking assay following the respective manufacturer's instructions. Specimens negative by EIA that fell within a gray zone 30% below the cutoff and negative cultures with one or more corresponding positive EIA results were tested further by cytocentrifugation and direct immunofluorescent assay. The overall sensitivity, specificity, positive predictive value, and negative predictive value for Syva versus culture were 94, 98.8, 85.5 and 99.6%, respectively. After resolution, the results were 94.5, 99.6, 94.5, and 99.6%, respectively. The parallel results for the Sanofi Microplate EIA versus culture were 94.0, 98.7, and 83.9, and 99.6%, respectively, and after being resolved, the results were 94.9, 100, 100, and 99.6%, respectively. In the small male population tested, the resolved results of the Sanofi Microplate EIA versus culture demonstrated sensitivity, specificity, positive predictive value, and negative predictive value of 100, 100, 100, and 100%, respectively. The present study demonstrated that the Sanofi Microplate EIA shortened protocol is highly sensitive and specific in comparison with cell culture and the Syva MicroTrak II EIA.
Performance of a rapid, on-site human immunodeficiency virus antibody assay in a public health setting
Authors: Kassler W J , Haley C , Jones W K , Gerber A R , Kennedy E J , George J R
Published: January 2020

Rapid, on-site human immunodeficiency virus (HIV) testing has the potential to improve the delivery of prevention services in publicly funded counseling and testing sites. The Single Use Diagnostic System (SUDS) HIV-1 is the only rapid enzyme immunoassay (EIA) approved for diagnostic use in the United States. To evaluate the feasibility of using SUDS in public clinics and to validate the test's performance in a public health laboratory, we conducted blinded SUDS testing on plasma sent for HIV testing. From 19 March through 30 June 1993, 1,923 consecutive samples from a sexually transmitted diseases clinic and an HIV counseling and testing clinic were tested on site with SUDS. Tests done in the first two weeks with a malfunctioning centrifuge n = 402) and those done when there were excessively high temperatures in the laboratory (n = 53) were analyzed separately. Of 1,466 tests, 39 were positive by both SUDS and EIA (with Western blot [immunoblot] confirmation) and 7 were SUDS positive and EIA negative. Western blotting was used as the "gold standard" to adjudicate these discrepancies. There were no SUDS-negative and EIA-positive tests. Compared with that of EIA (with Western blot confirmation), the sensitivity of SUDS was 100% (95% confidence interval, 88.8 to 100%) and the specificity was 99.5% (95% confidence interval, 98.9 to 99.8%). The positive predictive value of SUDS was 88% in the STD clinic and 81% in the HIV counseling and testing clinic. There was a 7.7-fold increase in false positives, from 0.48 to 3.7%, when there was inadequate centrifugation and when the temperature exceeded the manufacturer's recommendations. Rapid, on-site HIV testing by the SUDS assay is feasible and practical in public health settings. The test can be performed accurately, at reasonable cost, and within the time frame of a typical clinic visit. Caution should be used, however, as two conditions adversely affected the accuracy of this test: inadequate specimen preparation and elevated temperature.
Molecular probes for diagnosis of fungal infections
Authors: Sandhu G S , Kline B C , Stockman L , Roberts G D
Published: January 2020

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.
Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes
Authors: Rijpkema S G , Molkenboer M J , Schouls L M , Jongejan F , Schellekens J F
Published: January 2020

We developed a rapid and reliable method for the identification Borrelia burgdorferi sensu lato species in ticks. We used the DNA sequence polymorphism of the spacer region between 5S and 23S rRNA genes, which has been shown to be able to discriminate between eight genomic groups of B. burgdorferi sensu lato (D. Postic, M. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). Spacer DNA was amplified by PCR and was then hybridized to five membrane-bound oligonucleotides. The oligonucleotides were specific for B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and group VS116. A probe which reacted with all genomic groups of B. burgdorferi sensu lato was also used. Ninety-six ticks collected in the field were destructed by bead beating, and the supernatant was used directly in a PCR. B. burgdorferi sensu lato DNA was detected in 6 of 57 adult ticks (11%) and 9 of 39 nymphs (23%). B. garinii was found in three nymphs and four adults, three nymphs carried B. afzelii, and one adult and one nymph carried group VS116. Double infections with B. afzelii and group VS116 were found in two nymphs and one adult. Thus, our method can simultaneously identify three genomic groups of B. burgdorferi sensu lato in ticks collected in the field. This technique provides new ways to study the association of genomic groups present in ticks and the risk of Lyme borreliosis.
Comparison of three staining methods for detecting microsporidia in fluids
Authors: Didier E S , Orenstein J M , Aldras A , Bertucci D , Rogers L B , Janney F A
Published: January 2020

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEM-negative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.
Differential spirochetal infectivities to vector ticks of mice chronically infected by the agent of Lyme disease
Authors: Shih C M , Liu L P , Spielman A
Published: January 2020

We determined whether the infectivity of the Lyme disease spirochete (Borrelia burgdorferi) to vector ticks varies with the duration of infection in laboratory mice. Thus, noninfected nymphal deer ticks were permitted to feed on two strains of early (2 months after infection) and late (8 months after infection) spirochete-infected mice. The attached ticks were removed from their hosts at specified time intervals and were thereafter examined for spirochetes by direct immunofluorescence microscopy. Spirochetes can be acquired by nymphal ticks as fast as 8 h after attachment. More than 80% of the attached ticks acquired spirochetal infection within 48 h after feeding on early spirochete-infected mice. In contrast, spirochetal infectivity to ticks was less than 50% after feeding on late spirochete-infected mice. The overall infectivity of spirochete-infected mice to ticks correlated with the duration of tick attachment. In addition, there was no adverse effect on the spirochetal infectivity to ticks by high levels of host antibody against spirochetes, and no obvious differences in infectivity to ticks was observed by the site of tick feeding. We conclude that the span of spirochetal infectivity to ticks varies with the duration of infection in mice and suggest that spirochetes may persist and may be evenly distributed in the skin of infected hosts, regardless of prominent host immunity.
Clonal relatedness of Shiga-like toxin-producing Escherichia coli O101 strains of human and porcine origin
Authors: Franke S , Harmsen D , Caprioli A , Pierard D , Wieler L H , Karch H
Published: January 2020

Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans. In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors. The slt genes of five E. coli O101 strains were cloned and sequenced. For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs. The A subunit revealed more than 99% homology to that of SLT-IIe. DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates. None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E. coli hemolysin). The absence of virulence factors typical of SLT-I- and SLT-II-producing E. Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E. coli, suggests a new pathogenic mechanism for E. coli O101 infection of humans. For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases.
Identification and antimicrobial resistance patterns of clinical isolates of Clostridium clostridioforme, Clostridium innocuum, and Clostridium ramosum compared with those of clinical isolates of Clostridium perfringens
Authors: Alexander C J , Citron D M , Brazier J S , Goldstein E J
Published: January 2020

Clostridium ramosum, C. innocuum, and C. clostridioforme are frequently isolated from clinical specimens including blood. Because of Gram stain variability, a lack of spores, and atypical colonial morphology, identification of these species is often difficult. Three anaerobe identification kits were evaluated for their abilities to identify these species. For comparison, 11 strains of C. perfringens were evaluated in parallel. By using profile numbers and codebooks, the correct genus and species were identified, as follows: with the RapID ANA II kit, 100% (20 of 20) of C. ramosum isolates, 24% (5 of 21) of C. innocuum isolates, and 50% (10 of 20) of C. clostridioforme isolates; with the AnIDent kit, 60% (12 of 20) of C. ramosum isolates, 28% (6 of 21) of C. innocuum isolates, and 90% (18 of 20) of C. clostridioforme isolates; with the ATB32A kit, 70% (14 of 20) of C. ramosum isolates, 0% (0 of 21) of C. innocuum isolates, and 40% (8 of 20) of C. clostridioforme isolates. Profile numbers that overlapped several species were obtained as follows: with the RapID ANA II kit, 0% of C. ramosum isolates, 76% of C. innocuum isolates, and 40% of C. clostridioforme isolates; with the AnIDent kit 40% of C. ramosum isolates, 62% of C. innocuum isolates, and 5% of C. clostridioforme isolates; with the ATB32A kit, 15% of C. ramosum isolates, 52% of C. innocuum isolates, and 25% of C. clostridioforme isolates. One strain of C. innocuum was misidentified by the AnIDent kit, and the remainder yielded profile numbers that were not listed in the codebooks. The MICs of 11 antimicrobial agents including penicillin G, metronidazole, clindamycin, cefoxitin, cefotetan, imipenem, meropenem, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, and vancomycin were determined by the agar dilution method. All C. perfringens strains were susceptible to all antimicrobial agents tested. Various levels of resistance to cefoxitin, cefotetan, and penicillin G were noted with C. ramosum, C. clostridioforme, and C. innocuum. In addition, resistance to clindamycin was noted with C. ramosum (5%) and C. innocuum (10%). Most strains of C. innocuum were only moderately susceptible to vancomycin (MIC at which 90% of strains are inhibited, 4 micrograms/ml).
Enhanced specificity of truncated transmembrane protein for serologic confirmation of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by western blot (immunoblot) assay containing recombinant envelope glycoproteins
Authors: Varma M , Rudolph D L , Knuchel M , Switzer W M , Hadlock K G , Velligan M , Chan L , Foung S K , Lal R B
Published: January 2020

Immunoassays based on the highly immunogenic transmembrane protein of human T-cell lymphotropic virus type 1 (HTLV-1) (protein 21c) are capable of detecting antibodies in all individuals infected with HTLV-1 and HTLV-2. However, because of antigenic mimicry with other cellular and viral proteins, such assays also have a large proportion of false-positive reactions. We have recently identified an immunodominant epitope, designated GD21-I located within amino acids 361 to 404 of the transmembrane protein, that appears to eliminate such false positivity. This recombinant GD21-I protein was used in conjunction with additional recombinant HTLV type-specific proteins and a whole virus lysate to develop a modified Western blot (immunoblot) assay (HTLV WB 2.4). The sensitivity and specificity of this assay were evaluated with 352 specimens whose infection status was determined by PCR assay for the presence or absence of HTLV-1/2 proviral sequences. All HTLV-1-positive (n = 102) and HTLV-2-positive (n = 107) specimens reacted with GD21-1 in the HTLV WB 2.4 assay, yielding a test sensitivity of 100%. Furthermore, all specimens derived from individuals infected with different viral subtypes of HTLV-1 (Cosmopolitan, Japanese, and Melanesian) and HTLV-2 (IIa0, a3, a4, IIb1, b4, and b5) reacted with GD21-I in the HTLV WB 2.4 assay. More importantly, HTLV WB 2.4 analysis of 81 PCR-negative specimens, all of which reacted to recombinant protein 21e in the presence or absence of p24 and p19 reactivity in the standard WB assay, showed that only two specimens retained reactivity to GD21-I, yielding an improved test specificity for the transmembrane protein of 97.5%. None of 41 specimens with gag reactivity only or 21 HTLV-negative specimens demonstrated reactivity to GD21-I. In an analysis of additional specimens (n = 169) from different geographic areas for which PCR results were not available, a substantial increase in the specificity of GD21-I detection was demonstrated, with no effect on the sensitivity of GD21-I detection among specimens from seropositive donors. Thus, the highly sensitive, GD21-I-based HTLV WB 2.4 assay eliminates the majority of false-positive transmembrane results, thereby increasing the specificity for serologic confirmation of HTLV-1 and HTLV-2 infections.
Protection against Ehrlichia equi is conferred by prior infection with the human granulocytotropic Ehrlichia (HGE agent)
Authors: Barlough J E , Madigan J E , DeRock E , Dumler J S , Bakken J S
Published: January 2020

A Thoroughbred filly that developed clinical signs of equine granulocytic ehrlichiosis following inoculation with the human granulocytotropic ehrlichia was shown to be resistant to challenge with Ehrlichia equi, a closely related agent. This result further substantiates the close and potentially conspecific relationship between these two granulocytotropic ehrlichiae.
Case report of Streptomyces endocarditis of a prosthetic aortic valve
Authors: Mossad S B , Tomford J W , Stewart R , Ratliff N B , Hall G S
Published: January 2020

We describe the first case of prosthetic valve endocarditis due to a Streptomyces sp. The patient presented with fever, cutaneous embolic lesions, and bacteremia 3 months after aortic valve replacement. Treatment required valve replacement and a long course of parenteral imipenem.
Frozen stored murine hybridoma cells can be used to determine the virulence of Listeria monocytogenes
Authors: Bhunia A K , Westbrook D G , Story R , Johnson M G
Published: January 2020

Murine hybridoma cells, designated Ped-2E9, when stored up to 60 days at -196 degrees C or up to 48 days at -80 degrees C, gave results equivalent to those for freshly grown murine hybridoma cells in an in vitro pathogenicity assay of Listeria species. Thus, laboratories do not need to have their own tissue culture facilities to maintain the hybridoma cells for the assay described.
Comparison of Crystal Enteric/Nonfermenter system, API 20E system, and Vitek AutoMicrobic system for identification of gram-negative bacilli
Authors: Robinson A , McCarter Y S , Tetreault J
Published: January 2020

A comparative evaluation of the Crystal Enteric/Nonfermenter system (Crystal; Becton Dickinson, Cockeysville, Md.), API 20E (API; bioMérieux Vitek, Inc., Hazelwood, Mo.), and the Vitek GNI card (Vitek; bioMérieux Vitek) was performed with 512 clinical isolates of gram-negative bacilli, including 381 members of the family Enterobacteriaceae and 131 nonenteric bacilli. With supplemental testing, API, Crystal, and Vitek correctly identified to the genus and species level 505 (98.6%), 489 (95.5%), and 494 (96.5%) of the 512 isolates, respectively. Supplemental testing, as specified by the manufacturer, was required to identify 119 (23.2%), 18 (3.5%), and 5 (1.0%) of the isolates with the three systems, respectively. Of the 381 isolates from the family Enterobacteriaceae, API and Crystal correctly identified 90.3 and 91.6% by 18 to 24 h without supplemental testing, respectively, and Vitek identified 92.4 and 96.1% following 10 and 18 h of incubation, respectively. Of the 131 nonenteric organisms, API and Crystal correctly identified 28.2 and 93.9% by 18 to 24 h without supplemental testing, respectively, and Vitek identified 84.0% by 10 h and 93.9% by 18 h. Errors in identification with each system were infrequent and appeared to be randomly distributed among the genera evaluated. The three systems were comparable in accuracy when either a weighted clinical laboratory profile of organisms or a group of selected isolates in a stress test sample was evaluated (P > 0.05). There were no significant differences between the three systems in their ability to identify either the isolates in the weighted group or those in the stress test (P > 0.05). Crystal compared favorably with API and Vitek, which have established track records in clinical laboratories, and is acceptable for the identification of members of the Enterobacteriaceae and nonenteric bacilli in a clinical microbiology laboratory.
Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells
Authors: Upton S J , Tilley M , Brillhart D B
Published: January 2020

Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and antibiotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed free of debris, fresh cell culture media containing select supplements were added and cultures were reincubated. Parasite growth was assessed 66 h later by counting the number of parasite developmental stages in 25 random x 100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, folic acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro. The addition of insulin and the sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with any of the vitamins. Using the above information, we developed a supplemental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 micrograms of ascorbic acid, 1.0 micrograms of folic acid, 4.0 micrograms of 4-aminobenzoic acid, 2.0 micrograms of calcium pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 micrograms of streptomycin, and 0.25 microgram of amphotericin B (Fungizone) per ml (pH 7.4). The growth of c. parvum in this medium was found to be enhanced approximately 10-fold compared with that in control medium without additional glucose, insulin, or vitamins.
Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein
Authors: Bölin I , Lönroth H , Svennerholm A M
Published: January 2020

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.
Comparison of four different methods for detection of Cryptosporidium species
Authors: Kehl K S , Cicirello H , Havens P L
Published: January 2020

Newly available assays offer alternatives to conventional microscopic examination for Cryptosporidium spp. We compared two enzyme immunoassays, ProSpect Cryptosporidium microtiter assay (Alexon, Inc., Mountain View, Calif.) and Color Vue Cryptosporidium assay (Serady, Indianapolis, Ind.), and a direct immunofluorescent assay, Merifluor Cryptosporidium kit (Meridian Diagnostics, Cincinnati, Ohio), with acid-fast Kinyoun-staining for the detection of Cryptosporidium spp. Examinations were performed on 129 stool specimens received from patients during a recent waterborne outbreak. A specimen was considered positive when organisms could be identified visually by acid-fast and immunofluorescent stains or if organisms could be visualized by either acid-fast or immunofluorescent stain and detected by both enzyme immunoassays. The final number of positive specimens was 55. No single procedure detected all 55 positive specimens. Of these, ProSpect and Color Vue detected 52 (sensitivity, 94.5%), and the Kinyoun stain and Merifluor detected 53 (sensitivity, 96.4%). The final number of negative specimens was 74. One false-positive result was seen with both the Kinyoun stain and the ProSpect assay. The Color Vue and ProSpect assays required the most hands-on technologist time. The ProSpect assay and Merifluor kit were easiest to perform. The acid-fast stain was difficult to interpret. The Merifluor kit was easiest to read and was adaptable to both batch and single testing. Overall, the Kinyoun stain and the Merifluor test were preferable to both of the enzyme immunoassays because of the high reagent cost and hands-on time required for the enzyme immunoassays. The difficult interpretation of the Kinyoun stain smears made the Merifluor a more desirable test despite its higher cost. We conclude that all methods tested were equally sensitive and specific for the detection of Cryptosporidium spp. Ease of use, adaptability to batch testing, and cost are important criteria in determining the method of choice.
Immunoblot interpretation criteria for serodiagnosis of early Lyme disease
Authors: Engstrom S M , Shoop E , Johnson R C
Published: January 2020

We monitored the antibody responses of 55 treated patients with early Lyme disease and physician-documented erythema migrans. Six sequential serum samples were obtained from patients before, during, and until one year after antibiotic therapy and analyzed by in-house enzyme-linked immunosorbent (ELISA) and immunoblot assays. An immunoblot procedure utilizing a gradient gel and an image analysis system was developed. A relational database management system was used to analyze the results and provide criteria for early disease immunoblot interpretation. Recommended criteria for the immunoglobulin M (IgM) immunoblot are the recognition of two of three proteins (24, 39, and 41 kDa). The recommended criteria for a positive IgG immunoblot are the recognition of two of five proteins (20, 24 [> 19 intensity units], 35, 39, and 88 kDa). Alternatively, if band intensity cannot be measured, the 22-kDa protein can be substituted for the 24-kDa protein with only a small decrease in sensitivity. Monoclonal antibodies were used to identify all these proteins except the 35-kDa protein. With the proposed immunoblot interpretations, the sequential serum samples were examined. At visit 1, the day of diagnosis and initiation of treatment, 54.5% of the serum samples were either IgM or IgG positive. The peak antibody response, with 80% of the serum samples positive, occurred at visit 2, 8 to 12 days into treatment. The sensitivities of the IgM and IgG immunoblot for detecting patients that were seropositive into the study period were 58.5 and 54.6%, respectively, at visit 1 and 100% at visit 2. Twenty percent of the patients remained seronegative throughout the study. The specificities of the IgM and IgG immunoblots were 92 to 94% and 93 to 96%, respectively. The IgM immunoblot and ELISA were similar in sensitivities, whereas the IgG immunoblot had greater sensitivity than the IgG ELISA (P = 0.006).
Specific amplification of Rickettsia japonica DNA from clinical specimens by PCR
Authors: Furuya Y , Katayama T , Yoshida Y , Kaiho I
Published: January 2020

The gene encoding the 17,000-molecular-weight genus-common antigen (17K genus-common antigen) has been cloned and sequenced from Rickettsia japonica. The primer pair used for PCR was designed from this sequence. A 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and also the DNA from blood clots of patients with spotted fever group rickettsiosis. The results indicated that this method is suitable for the diagnosis of spotted fever group rickettsiosis in Japan.
Oligotrophic bacteria isolated from clinical materials
Authors: Tada Y , Ihmori M , Yamaguchi J
Published: January 2020

Oligotrophic bacteria (oligotrophs) are microorganisms that grow in extremely nutritionally deficient conditions in which the concentrations of organic substances are low. Many oligotrophic bacteria were isolated from clinical materials including urine, sputum, swabbings of the throat, vaginal discharges, and others. Seventy-seven strains of oligotrophic bacteria from 871 samples of clinical material were isolated. A relatively higher frequency of isolation of oligotrophic bacteria was shown in drainage, sputum, and throat specimens. Eleven strains of the obligate oligotrophic bacteria recovered showed scant growth on enriched medium, blood agar, and nutrient agar. Oligotrophic bacteria were isolated from a variety of materials but were not found in routine bacteriologic examinations in the hospital laboratory. The clinical significance of such oligotrophic bacteria is uncertain.
Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations
Authors: Pane F , Buttò S , Gobbo M L , Franco M , Butteroni C , Pastore L , Maiorano G , Foggia M , Cataldo P T , Guarino A
Published: January 2020

We used a colorimetric polymerase chain reaction (PCR)-based assay in kit form to detect directly human immunodeficiency virus type 1 (HIV-1) proviral gag sequences in peripheral blood cells from 68 healthy blood donors, 51 subjects at risk for HIV infection, 122 patients with HIV-1 infection, 11 patients with indeterminate Western blot (immunoblot) results, 4 blood donors HIV-1 positive by enzyme immunoassay, and 13 children born to HIV-1-seropositive mothers. The results obtained in the blood donors and HIV-1-infected patients demonstrated the high degree of diagnostic specificity and sensitivity of the PCR method. HIV-1 infection was excluded in 10 of the 11 patients with indeterminate Western blot results and in all four enzyme immunoassay-positive blood donors. A diagnosis of HIV infection was ruled out by negative PCR results in 5 of 13 children from seropositive mothers, which excluded vertical transmission of the infection in these cases; these children were younger than 3 months and had positive serological results. Two at-risk patients with negative serological results had positive PCR results. All results were confirmed by conventional PCR. In conclusion, colorimetric PCR, which is commercially available in kit form, is an easy and reliable technique that can be used to detect proviral HIV-1 genomes in blood cells, and despite the limitations owing to HIV genome variability, it is useful in the clinical setting for the diagnosis of HIV infection in selected categories of patients.
Characterization of genetically distinct subgroup of Candida albicans strains isolated from oral cavities of patients infected with human immunodeficiency virus
Authors: McCullough M , Ross B , Reade P
Published: January 2020

During the course of a study of oral Candida albicans strains from 60 human immunodeficiency virus-infected patients over a 2.5-year period, 18 of the 295 C. albicans isolates had genomes that failed to hybridize with a C. albicans-specific DNA probe (27A). These strains were germ tube positive and chlamydospore positive and were identified as C. albicans by the ID 32C test (API Systems, Montlieu, France). These strains were analyzed for the presence of two other C. albicans-specific DNA segments by PCR. The first was a C. albicans 1,348-bp species-specific sequence, and the second was a 1,059-bp C. albicans repetitive element. The probe 27A-hybridizing strains yielded PCR products which differed from those of the nonhybridizing strains. Five of these genetically atypical C. albicans strains and 98 of the C. albicans strains were then analyzed for purported virulence factors. The genetically atypical C. albicans strains, in comparison with typical C. albicans strains, produced greater amounts of extracellular proteinase (P = 0.038, Student's t test), adhered to a greater degree to buccal epithelial cells (P = 0.018, Student's t test), and were less susceptible to the antifungal drug 5-flucytosine (P = 0.0003, Mann-Whitney test). Analysis of these strains with other common antifungal drugs showed no statistically significant variation in susceptibility. The results of this study indicated that these genetically atypical C. albicans strains possess increased virulence in comparison with typical C. albicans strains.
Techniques for controlling variability in gram staining of obligate anaerobes
Authors: Johnson M J , Thatcher E , Cox M E
Published: January 2020

Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes.
Agglutination of pYV+ Yersinia enterocolitica strains by agglutinin from Mangifera indica
Authors: Wauters G , Charlier J , Janssens M
Published: January 2020

Agglutination of 271 strains of Yersinia enterocolitica and related species grown at 37 degrees C by a 0.01% dilution of the agglutinin from Mangifera indica was correlated with the presence of the virulence plasmid. The study of YadA mutants suggested that the YadA protein is the target of the plant agglutinin.
Evaluation of a modified nitrous acid extraction latex agglutination kit for grouping beta-hemolytic streptococci and enterococci
Authors: Petts D N
Published: January 2020

The rapid and accurate identification of the Lancefield group of beta-hemolytic streptococci and enterococci is an important procedure in clinical laboratories. Latex agglutination techniques are more rapid and technically less demanding than traditional extraction-precipitation methods. Prolex (Pro-Lab Diagnostics, Richmond Hill, Ontario, Canada) is a latex agglutination kit which contains modified nitrous acid reagents to extract antigens and can be used to detect group D antigen in streptococci and enterococci, as well as group A, B, C, F, and G antigens. A total of 302 strains of streptococci and enterococci were tested with this kit. All streptococci of groups A (41 strains), B (39 strains), C (35 strains), D (3 strains), F (10 strains), and G (48 strains) were correctly grouped, as were 125 (97%) of 129 strains of enterococci. Prolex is a reliable method for grouping the beta-hemolytic streptococci and enterococci most frequently encountered in clinical laboratories.
Methods for predicting susceptibility of Streptococcus pneumoniae to cefixime
Authors: Barry A L , Fuchs P C
Published: January 2020

Among 698 Streptococcus pneumoniae isolates, 475 were penicillin susceptible and > 99% of those were susceptible to 0.5 microgram of cefixime per ml; other pneumococci were tentatively assumed to be resistant to cefixime. A 1-microgram oxacillin disk was more reliable than a 5-micrograms cefixime disk for predicting susceptibility to cefixime.
Diagnosis of American trypanosomiasis (Chagas' disease) by the new complement fixation test
Authors: Garcia E , Ramirez L E , Monteon V , Sotelo J
Published: January 2020

A new immunodiagnostic method of complement fixation was used for serodiagnosis of American trypanosomiasis; 92% sensitivity and 99% specificity were obtained, for an overall accuracy of 97%. This test can be used in field studies, obviating the use of most laboratory equipment and imported reagents; places where economic limitations hinder the use of other immunodiagnostic procedures; and in association with other tests for confirmation of the diagnosis.
Alterations in sample preparation increase sensitivity of PCR assay for diagnosis of chancroid
Authors: Johnson S R , Martin D H , Cammarata C , Morse S A
Published: January 2020

A PCR assay for the detection of Haemophilus ducreyi in clinical specimens taken from genital ulcers was developed. Although H. ducreyi, when present in such specimens, could be detected by PCR, the sensitivity of the assay was reduced by the presence of Taq polymerase inhibitors in the specimen. The sensitivity of the PCR assay was improved by the use of detergents in preparing nuclei acids from clinical specimens and by the inclusion of a dialysis step prior to amplification. In addition, sodium phosphate included in the transport medium was found to be an inhibitor of the Taq polymerase.
Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG
Authors: Frothingham R
Published: January 2020

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Seven strains of the M. tuberculosis complex were sequenced in a region of about 300 bp which contains multiple 15-bp tandem repeats and which is part of a 1,551-bp open reading frame. Four distinct sequences were obtained, each defining a sequevar. A sequevar includes the strain or strains with a given sequence. The type strain M. tuberculosis TMC 102 (H37Rv) was designated sequevar MED-G. When compared to MED-G, sequevar LONG had an insertion of one 15-bp tandem repeat and sequevar SHORT had a deletion of one tandem repeat. Sequevar MED-C had a G-->C substitution, coding for the conservative change Ser-->Thr. BanI cuts only sequevar MED-C at the site of the substitution. PCR-restriction enzyme analysis was used to determine the sequevars of 92 M. tuberculosis complex strains. All 23 M. bovis BCG strains belonged to sequevar MED-C. The M. africanum type strain was sequevar SHORT. The remaining 68 strains of M. tuberculosis, M. bovis (not BCG), and M. microti were sequevars LONG (3 strains) or MED-G (65 strains). PCR-restriction enzyme analysis was applied to reference strains and clinical isolates with a worldwide distribution. This method provides rapid, sensitive, and specific identification of the important vaccine strain M. bovis BCG.
Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein
Authors: Stucki U , Frey J , Nicolet J , Burnens A P
Published: January 2020

To facilitate discrimination between the closely related enteropathogens Campylobacter jejuni and C. coli, unique differences in antigenic surface structure were examined. A genomic library of C. jejuni 81116 was constructed in plasmid pBluescriptIISK- and expressed in Escherichia coli K-12. Rabbit hyperimmune serum raised against C. jejuni ATCC 29428 recognized a clone expressing a C. jejuni 24-kDa membrane-associated protein. Antiserum raised against sonicated recombinant E. coli expressing the 24-kDa protein reacted with C. jejuni, whereas C. coli did not react specifically. Determination of the nucleotide sequence of the DNA insert of this recombinant plasmid revealed an open reading frame encoding 214 amino acids; the gene was designated mapA; and its gene product was designated MAPA. The 18 N-terminal amino acid residues constitute a signal sequence characteristic of prokaryotic membrane lipoproteins. In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni were unequivocally discriminated from 126 other campylobacters, including 34 C. coli isolates. A PCR test based on the mapA sequence was developed for identification of C. jejuni. A PCR product was obtained with all of the clinical isolates of C. jejuni tested from human, dog, cat, bovine calf, and chicken sources. Recombinant MAPA with an added C-terminal six-histidine tail was affinity purified and used to immunize rabbits. The rabbit anti-MAPA serum specifically recognized the protein in whole cells of C. jejuni on Western blots (immunoblots). The MAPA protein was present in all of the C. jejuni strains tested and was absent in C. coli and related campylobacters.