bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus
Authors: Neville J A , Prescott L E , Bhattacherjee V , Adams N , Pike I , Rodgers B , El-Zayadi A , Hamid S , Dusheiko G M , Saeed A A , Haydon G H , Simmonds P
Published: January 2020

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.
Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp
Authors: Grundmann H J , Towner K J , Dijkshoorn L , Gerner-Smidt P , Maher M , Seifert H , Vaneechoutte M
Published: January 2020

Seven laboratories in six European countries examined 40 isolates belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex to investigate whether standardized protocols and quality-controlled reagents could produce reliable, discriminatory, and reproducible PCR-based fingerprinting results. Four PCR protocols with different primers (primers DAF4, ERIC-2, M13, and REP1 + REP2) were used. The epidemiological conclusions reached by the participating laboratories were substantially correct, with 96.4% of the total isolate grouping allocations agreeing with the consensus view. All laboratories identified the main epidemiological clusters, and each laboratory also identified two non-outbreak-related isolates. There were no significant differences between the isolate grouping results obtained by the different protocols and with the different primers. Visual comparison indicated that the standardized protocols and reagents yielded reproducible fingerprint patterns, but with some variations in particular band intensities. Minor variations in fingerprint profiles were detected, but computer-assisted analysis of PCR fingerprints obtained on agarose gels demonstrated that 88.3 to 91.6% (depending on the source of DNA) of the patterns clustered correctly, while 96.4 to 98.9% of the patterns clustered correctly following automated high-resolution laser fluorescence analysis. Correlation of the patterns for isogenic isolates ranged from 83.3 to 86.6% but was slightly better (mean correlation, 87.1%) for centrally prepared DNA extracts than for DNA extracts prepared by individual laboratories (mean correlation, 84.7%). It was concluded that independently produced PCR fingerprint patterns can be obtained reproducibly for Acinetobacter spp. at the practical level if (i) quality-controlled reagents, (ii) standardized extraction of DNA, and (iii) standardized amplification conditions are used.
Comparison of enzyme immunoassay, PCR, and type-specific cDNA probe techniques for identification of group A rotavirus gene 4 types (P types)
Authors: Masendycz P J , Palombo E A , Gorrell R J , Bishop R F
Published: January 2020

This study was designed to evaluate three techniques most commonly used to identify the VP4 (P) types of human group A fecal rotaviruses. The techniques included PCR with nested primers and hybridization with PCR-generated probes (to determine the P genotypes). The results obtained by these genetic techniques were evaluated against those obtained by an enzyme immunoassay (EIA) incorporating neutralizing monoclonal antibodies (N-MAbs) reacting with three major human P serotypes (serotypes P1A, P1B, and P2A). The P types of the rotaviruses present in 102 fecal specimens were determined under code by each of the three assays. The specificity of each assay was evaluated against a "gold standard" putative P type (P serotype and genotype) deduced from knowledge of the VP7 (G) type and the origin of the fecal specimen. Overall comparison of the results showed respective sensitivities and specificities of 92 and 92% for reverse transcription-PCR, 80 and 99% for hybridization, and 73 and 91% for EIA with N-MAbs. The hybridization assay retained high sensitivity with specimens stored for > or = 10 years. Hybridization assays with nonradioactive probes are relatively inexpensive and are suited for use in developing countries. In summary, both genetic assays showed high sensitivities and specificities in assigning a P type to human fecal rotavirus strains. Further evaluation of the EIA with N-MAbs is required, together with incorporation of new N-MAbs for the detection of the additional P types detected in developing countries.
Laboratory methods for detection of Chlamydia trachomatis: survey of laboratories in Washington State
Authors: Suchland K L , Counts J M , Stamm W E
Published: January 2020

The last decade has witnessed the development of a wide variety of diagnostic tests for Chlamydia trachomatis. In order to determine what laboratory methods are being used to detect C. trachomatis infections in Washington State and to identify factors influencing test selection, between April 1995 and October 1995 we conducted a mailed questionnaire survey of all 112 laboratories certified to do chlamydia testing. Of these, 20 had discontinued testing for C. trachomatis, and responses were obtained from 89 (97%) of the remaining 92 laboratories. Surprisingly, 38 (43%) of the 89 laboratories used rapid tests such as Clearview and Surecell, making such tests the most commonly used laboratory tests. Laboratories which used rapid tests had lower test volumes, less experience performing tests for C. trachomatis, less frequent attendance at professional meetings, and greater reliance on manufacturers for information compared with laboratories which used other methods. Confirmation of non-culture-positive results was provided by 28 (34%) of the 82 laboratories doing non-culture-based tests. Forty-one (47%) of 88 laboratories reported having compared their method with another method. Test volume was the strongest predictor of laboratories which confirmed positive non-culture-based test results and which had performed a laboratory comparison of methods. We conclude that rapid tests for C. trachomatis are often being used inappropriately and that efforts are needed to improve effective implementation and quality assurance of laboratory testing for C. trachomatis.
Necrotizing pneumonia caused by Penicillium chrysogenum
Authors: D'Antonio D , Violante B , Farina C , Sacco R , Angelucci D , Masciulli M , Iacone A , Romano F
Published: January 2020

We report a case of necrotizing pneumonia due to Penicillium chrysogenum in a 57-year-old woman operated on for lung cancer. The residual right lower pulmonary lobe was infiltrated by Penicillium chrysogenum. The patient underwent a second pulmonary right lobectomy and was successfully treated with oral itraconazole. To our knowledge, this is the first case of pneumonia due to P. chrysogenum.
Detection of Chlamydia trachomatis in urine samples by nucleic acid tests: comparison with culture and enzyme immunoassay of genital swab specimens
Authors: Schepetiuk S , Kok T , Martin L , Waddell R , Higgins G
Published: January 2020

Two commercially available nucleic acid-based tests, ligase chain reaction (LCR; Abbott Laboratories) and PCR (Roche Diagnostics), for the detection of Chlamydia trachomatis in male and female urine samples were compared with culture and enzyme immunoassay (EIA) (Microtrak; Syva) for C. trachomatis detection in genital samples. The samples were collected from 1,005 patients who attended a sexually transmitted disease clinic. In this study population, the prevalence of the infection was 4%. Specimens which were reactive in any of the tests were retested with a different PCR test using primers directed against the major outer membrane protein gene. With a "gold standard" of a positive culture, or any other positive test result if it was confirmed by an independent test, the Roche PCR (95% sensitive, 99.9% specific) was more sensitive than the LCR (75% sensitive, 100% specific) (chi2, P < 0.0001) while both tests were more sensitive than culture (58% sensitive, 100% specific) or EIA (45% sensitive, 100% specific) (chi2, P < 0.001). The Roche PCR and Abbott LCR tests of urine identified 65% and 30% more positive patients, respectively, than did testing by culture of urethral or cervical specimens. Nucleic acid testing of urine specimens for C. trachomatis is a more sensitive and convenient method for the detection of genital infection.
Evaluation of a new enzyme immunoassay based on recombinant Rubella virus-like particles for detection of immunoglobulin M antibodies to Rubella virus
Authors: Grangeot-Keros L , Enders G
Published: January 2020

A new enzyme immunoassay for the detection of specific antibodies to rubella virus was evaluated at two different sites. This assay, the Roche Cobas Core Rubella IgM EIA recomb, uses a recombinant rubella virus-like particle and is based upon the immunoglobulin M (IgM) capture principle. It was compared to the Abbott IMx Rubella IgM test and to the Sorin ETI-RUBEK-M reverse test. The relative clinical specificities were 99.30% for the Roche test, 98.26% for the Abbott test, and 100% for the Sorin test. The relative clinical sensitivities were 100, 93.87, and 82.65%, respectively. In the case of most primary infections, IgM antibodies could be detected immediately at the onset of the disease and for up to 7 weeks. In the case of vaccinations, they could be detected between 3 and 12 weeks after vaccination.
Comparison of manual Amplicor PCR, Cobas Amplicor PCR, and LCx assays for detection of Chlamydia trachomatis infection in women by using urine specimens
Authors: Pasternack R , Vuorinen P , Pitkäjärvi T , Koskela M , Miettinen A
Published: January 2020

We compared the Roche Amplicor PCR, Roche Cobas Amplicor PCR, and Abbott LCx assays by using urine specimens for the detection of Chlamydia trachomatis infections in a female population. First-catch urine and endocervical swab specimens were collected from a total of 442 patients. Urine specimens were tested by the manual Roche Amplicor PCR, the automatic Roche Cobas Amplicor PCR, and the Abbott LCx assays as instructed by the manufacturers. For the Cobas Amplicor PCR, the internal control protocol was used for every specimen to reveal the presence of polymerase inhibitors. Cell culture of cervical specimens was used as a reference method. Of 442 patients, 50 (11.3%) were confirmed to have chlamydial infection. The diagnostic sensitivity and specificity of cell culture with cervical swab specimens were 88 and 100%, respectively. With urine specimens the sensitivity and specificity for the manual Amplicor PCR assay were 100 and 99.7%, respectively; those for the automatic Cobas Amplicor PCR assay were 94 and 99.2%, respectively; and those for the LCx assay were 94 and 100%, respectively. Thus, all amplification methods with urine specimens proved to be highly sensitive and specific for the detection of C. trachomatis infection in women. No statistically significant differences in the test performances could be demonstrated for specimens from this population. All three amplification techniques with urine specimens proved to be superior to cell culture with cervical swab specimens in diagnosing C. trachomatis infections in women.
Genetic and phenotypic characterization of intestinal spirochetes colonizing chickens and allocation of known pathogenic isolates to three distinct genetic groups
Authors: McLaren A J , Trott D J , Swayne D E , Oxberry S L , Hampson D J
Published: January 2020

Infection with intestinal spirochetes has recently been recognized as a cause of lost production in the poultry industry. Little is known about these organisms, so a collection of 56 isolates originating from chickens in commercial flocks in Australia, the United States, The Netherlands, and the United Kingdom was examined. Strength of beta-hemolysis on blood agar, indole production, API ZYM enzyme profiles, and cellular morphology were determined, and multilocus enzyme electrophoresis was used to analyze the extent of genetic diversity among the isolates. The results were compared with those previously obtained for well-characterized porcine intestinal spirochetes. The chicken isolates were genetically heterogeneous. They were divided into 40 electrophoretic types distributed among six diverse genetic groups (groups b to g), with a mean genetic diversity of 0.587. Strains in two groups (groups d and e) may represent new species of Serpulina, and the groups contained only strains isolated from chickens. Three genetic groups contained isolates previously shown to be pathogenic for chickens. These corresponded to the proposed species "Serpulina intermedius," to an unnamed group (group e), and to Serpulina pilosicoli. Two of the chicken isolates (one "S. intermedius" and one S. pilosicoli isolate) were strongly beta-hemolytic, two (both "S. intermedius") had an intermediate level of beta-hemolysis, and the rest were weakly beta-hemolytic. Fourteen isolates of "S. intermedius" produced indole, as did one isolate from group d. Isolates identified as S. pilosicoli resembled porcine isolates of this species, having four to six periplasmic flagella inserted subterminally in a single row at each end of the cell, and had tapered cell ends. All other spirochetes were morphologically similar, having seven or more periplasmic flagella and blunt cell ends. The identification of three genetic groups containing pathogenic isolates provides an opportunity for more detailed epidemiologic studies with these pathogens and for the development of improved diagnostic tests.
Clinical and molecular study of Corynebacterium diphtheriae systemic infections in France. Coryne Study Group
Authors: Patey O , Bimet F , Riegel P , Halioua B , Emond J P , Estrangin E , Dellion S , Alonso J M , Kiredjian M , Dublanchet A , Lafaix C
Published: January 2020

Diphtheria is a disease with a long history that almost completely disappeared from developed countries. In addition, until 1987, systemic infections involving Corynebacterium diphtheriae were rare. However, in 1990, an epidemic occurred in Russia. These two circumstances have provided the stimulus to gain insight into the situation in France. In fact, between 1987 and 1993, a total of 59 C. diphtheriae strains were isolated. Epidemiological data were collected for patients from whom 40 strains were isolated from normally sterile sites, including 34 from blood cultures, and half of the bacteremic patients developed endocarditis. Osteoarticular involvement was noted in 11 of these 40 patients, including 5 bacteremic patients. The fatality rate following bacteremia was 36%, despite specific antibiotic treatment (beta-lactams and aminoglycosides). The mean age of the participants was 38 years, with half of the patients subsisting under low socioeconomic conditions and suffering from homelessness or alcoholism. Apparently, the skin turned out to be the major route of transmission in this reemerging disease. Eighty-eight percent of the isolates belonged to the C. diphtheriae biotype mitis. These were found predominantly in the Paris area, and most were of the same ribotype. Those isolates originating from the overseas territories (Guyana and New Caledonia) belonged to C. diphtheriae biotype gravis. No strains were positive for the tox gene by PCR. This study attests to the persistent circulation in France of C. diphtheriae in the form of systemic infections. The matter is especially significant since these strains are nontoxigenic and are of a unique ribotype. The strains are, however, sensitive to most antibiotics, although 20% are rifampin resistant.
Concordance of Porphyromonas gingivalis colonization in families
Authors: Tuite-McDonnell M , Griffen A L , Moeschberger M L , Dalton R E , Fuerst P A , Leys E J
Published: January 2020

Periodontitis is a widespread disease that appears to be due to a specific bacterial infection. Several species of bacteria have been investigated as potential pathogens, and particularly strong evidence links the presence of Porphyromonas gingivalis with indicators of periodontitis. Information concerning the transmission of P. gingivalis between human contacts may be important in determining risk factors for disease and developing preventive strategies. A few small studies have provided some evidence of transmission between related individuals, but no large-scale study of families that would reflect the typical transmission of this pathogen in the population has been reported. The purpose of this study was to investigate the transmission of P. gingivalis within randomly selected, extended families. The colonization status of 564 members of multigeneration families was determined, and the degree of concordance observed among members of these families was then compared to that expected to occur based on the prevalence of colonization in the population studied. A PCR assay was used for detection of P. gingivalis. Concordance in colonization was more frequently observed within entire families (P = 0.0000) and for spouses (P < 0.001), children and their mothers (P < 0.001), children and their fathers (P < 0.01), adults and their mothers (P < 0.005), and siblings (P < 0.05) than would be expected if P. gingivalis were randomly distributed in the population studied. Results showed that contact with an infected family member substantially increased the relative risk of colonization in these intrafamilial pairs. This indicates that P. gingivalis is commonly transmitted by contact with an infected family member.
Identification and characterization of Serpulina pilosicoli isolates recovered from the blood of critically ill patients
Authors: Trott D J , Jensen N S , Saint Girons I , Oxberry S L , Stanton T B , Lindquist D , Hampson D J
Published: January 2020

The phenotypic and genetic characteristics of spirochetes isolated from the blood of one U.S. and six French patients with severe clinical disease or impaired immunity were examined. All spirochetes were anaerobic, weakly beta-hemolytic, positive for hippurate hydrolysis, and negative for beta-glucosidase activity. Cell lengths ranged from 4 to 8 microm, and each isolate had between 8 and 12 periplasmic flagella per cell. These features were consistent with the spirochetes' being Serpulina pilosicoli, the agent of intestinal spirochetosis. All isolates were positive in a PCR assay amplifying a portion of the S. pilosicoli 16S rRNA gene, and they all grouped with fecal isolates of S. pilosicoli in multilocus enzyme electrophoresis (MLEE). The blood isolates could be differentiated from each other by MLEE, although the U.S. and two French isolates were closely related. Apparently S. pilosicoli may translocate from the large intestine to establish spirochetemia. The clinical significance of this finding remains uncertain and requires further investigation.
Epidemiology of group C rotavirus infection in Western New York women of childbearing age
Authors: Riepenhoff-Talty M , Morse K , Wang C H , Shapiro C , Roberts J , Welter M , Allen M , Evans M J , Flanagan T D
Published: January 2020

Umbilical cord serum samples (380), an average of 10 per month for 3 years (1990 to 1992), were tested by indirect immunofluorescence assay for group C rotavirus immunoglobulin G. Thirty percent were positive, suggesting that approximately one-third of women of childbearing age in western New York have experienced group C rotavirus infection.
Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples
Authors: Schwab K J , Estes M K , Neill F H , Atmar R L
Published: January 2020

Norwalk virus (NV) and the Norwalk-like viruses are important human pathogens that cause epidemic acute viral gastroenteritis. Current techniques used to recover NV from clinical samples involve multistep viral extraction and elution procedures with subsequent viral detection by reverse transcription-PCR (RT-PCR). In this study, a simple method using heat to recover viral RNA from 45 stool samples was compared to a conventional viral RNA extraction technique, with subsequent analysis by RT-PCR. In addition, we used an internal RNA standard for the detection of inhibitors present in processed samples. Our results indicate that the use of heat to recover NV RNA from stool samples has a sensitivity for the detection of NV RNA that is similar to the more labor-intensive, time-consuming, conventional RNA extraction technique. The use of an RNA internal standard permits the detection of inhibitors present in processed samples, allowing the identification of false negatives. The standard we developed has the advantage of allowing differential detection between wild-type viral RNA and standard using internal oligoprobe hybridization.
Evaluation of a quantitative competitive PCR assay for measuring herpes simplex virus DNA content in genital tract secretions
Authors: Hobson A , Wald A , Wright N , Corey L
Published: January 2020

Previous studies have shown an association between the approximate titer of herpes simplex virus (HSV) DNA in clinical specimens and the ability to isolate HSV from genital secretions. To control for variance in amplification conditions, we developed a competitive quantitative PCR (QC PCR) for the detection of HSV DNA. The assay accurately measured from 10 to 10(6) copies of HSV DNA. We compared the QC PCR with our previous semiquantitative detection method and found concordance for 61 of 63 positive specimens. We also evaluated the HSV DNA content from individual swabs of genital secretions obtained from individual sites of the genital tract (cervix, vulva, and rectum) with that from one swab with secretions from all three sites. The concordance for detecting HSV DNA was 91%; for only 4 of 143 collection days was there a > 1 log difference between the two collection methods. A single swab with secretions from all three genital sites and evaluated in a QC PCR format can accurately measure the frequency of subclinical and clinical shedding of HSV and the titer of HSV shed from the genital region. Such an approach should be very useful in the evaluation of antiviral chemotherapy for HSV.
Evaluation of the recombinant 38-kilodalton antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent
Authors: Wilkinson R J , Hasløv K , Rappuoli R , Giovannoni F , Narayanan P R , Desai C R , Vordermeier H M , Paulsen J , Pasvol G , Ivanyi J , Singh M
Published: January 2020

The diagnosis of infection caused by Mycobacterium tuberculosis is of increased public health concern following increases in the number of cases in developed countries and major increases in developing countries associated with the spread of human immunodeficiency virus (HIV) infection. The specificity of purified protein derivative skin testing for the detection of infection is compromised by exposure to environmental mycobacteria. Examination of sputum detects the most infectious patients, but not those with extrapulmonary disease. The 38-kDa antigen of M. tuberculosis contains two M. tuberculosis-specific B-cell epitopes. We overexpressed the gene for this antigen in Escherichia coli and evaluated the recombinant product in in vitro assays of T-cell function and as a target for the antibody response in humans. The sensitivity and specificity of the antigen as a skin test reagent were also assessed in outbred guinea pigs. We found that 69% of healthy sensitized humans recognize the antigen in vitro, as manifested by both cell proliferation and the production of gamma interferon. Untreated patients initially have a lower frequency of response (38%); this recovers to 72% during therapy. A total of 292 patients (20 with HIV coinfection) and 58 controls were examined for production of antibody to the 38-kDa antigen by using a commercially available kit. The sensitivity of the test in comparison with that of culture was 72.6%, and the specificity was 94.9%. The antigen was also tested for its ability to induce skin reactions in outbred guinea pigs sensitized by various mycobacterial species. The antigen provoked significant skin reactions in M. tuberculosis-, M. bovis BCG-, and M. intracellulare-sensitized animals. The significance of these findings and the usefulness of this antigen in immunodiagnosis are discussed.
Screening for active toxoplasmosis in patients by DNA hybridization with the ABGTg7 probe in blood samples
Authors: Angel S O , Matrajt M , Margarit J , Nigro M , Illescas E , Pszenny V , Amendoeira M R , Guarnera E , Garberi J C
Published: January 2020

We report the potential use of a specific Toxoplasma gondii DNA probe (ABGTg7). We applied a dot blot hybridization assay to blood samples for the diagnosis of cerebral toxoplasmosis (CT), acute toxoplasmic lymphadenopathy (ATL), and disseminated toxoplasmosis in transplant recipients (TRs). We studied a total of 84 individuals: 38 patients and 46 controls. We found positive hybridization signals for 12 (66.7%) of 18 patients with confirmed CT, 9 (52.9%) of 17 patients with ATL, and 2 (66.7%) of 3 TRs. PCR assays were performed in parallel for patients with ATL, resulting in T. gondii DNA detection for 10 patients (58.8%). A comparative study between dot blot and PCR assays performed with the blood of mice that had been experimentally infected with tachyzoites gave similar results: 60 and 70% positive results, respectively. Finally, the sum of positive values obtained by both DNA tests (dot blot assay plus PCR) increased the rate of positivity for ATL patients to 76.4%. These results demonstrate that the T. gondii ABGTg7 repetitive DNA element is an additional useful resource for diagnosing Toxoplasma parasitemia in patients with CT and ATL and in TRs. Thus, our ABGTg7-based dot blot test may lead to an improvement in T. gondii detection methods in patients with acute toxoplasmosis.
Comparison of the western blot assay with the neutralizing-antibody and enzyme-linked immunosorbent assays for measuring antibody to verocytotoxin 1
Authors: Reymond D , Karmali M A , Clarke I , Winkler M , Petric M
Published: January 2020

A Western blot (immunoblot) assay (WBA) was developed to detect immunoglobulin G (IgG) antibodies against Escherichia coli Verocytotoxin 1 (VT1) by using a chemiluminescence detection system. The assay was compared with a VT1-neutralizing-antibody (VT1-NAb) assay and an anti-VT1 IgG enzyme-linked immunosorbent assay (ELISA). When four human serum samples that were known to be positive by VT1-NAb assay and ELISA were titrated to the endpoint by the three assays, the WBA gave endpoint titers that were up to 8-fold higher than those by ELISA and up to 256-fold higher than those by the VT1-NAb assay. Of 32 serum samples that were known to be positive by VT1-NAb assay and ELISA, 31 (97%) were positive by WBA; the one sample with a discrepant result gave borderline results by the VT1-NAb assay and ELISA. Of 52 serum samples that were known to be negative by the VT1-NAb assay and ELISA, 50 (96%) were negative and 2 (4%) were positive by WBA. Of 44 serum samples that gave discrepant results by the VT1-NAb assay and ELISA, neither of the latter correlated with the results of WBA. In an investigation of 19 pairs of acute- and convalescent-phase serum samples from patients with hemolytic-uremic syndrome, 10 pairs that were positive by the VT1-NAb assay were also WBA positive, while 9 pairs that were NAb negative were also WBA negative. The WBA is inherently more specific and sensitive than either the NAb assay or the ELISA and may be used as a "gold standard" to detect IgG antibodies to VT1. Like the NAb assay and the ELISA for detecting antibodies to VT1, the WBA has little to offer in the diagnostic setting but is expected to play an important role in seroepidemiological studies.
Deterioration of detectable human immunodeficiency virus serum p24 antigen in samples stored for batch testing
Authors: Lathey J L , Marschner I C , Kabat B , Spector S A
Published: January 2020

Virologic measurements are becoming important surrogate markers for therapeutic efficacy in clinical trials with human immunodeficiency virus (HIV)-infected subjects. One such marker which is inexpensive and easily evaluated is the HIV p-24 antigen. To determine the storage stability of p24 antigen assayed by enzyme-linked immunosorbent assay of serum collected during clinical trials, a retrospective analysis was performed. The p24 antigen results were available from four Adult or Pediatric AIDS Clinical Trials Group protocols: studies 047, 050, 128, and 213. Paired samples (n = 930) which were assayed by ELISA for p24 antigen both in real time and in batch were analyzed for agreement. Batch and real-time values were correlated; however, there was a lack of agreement which increased with prolonged storage time of batched samples and greater p24 antigen levels. The p24 antigen values were significantly lower in the batched samples, which had a maximum storage time of 1,548 days. The degradation rate of p24 antigen per year was 0.052 log10 for samples with less than 30 pg/ml, 0.197 log10 for those with 30 to 100 pg/ml, and 0.245 log10 for those with > 100 pg/ml. Due to degradation over time, use of p24 antigen values from batch assays with long-term storage could bias study results toward a lack of treatment effect. On the basis of these results we make the following recommendations. (i) Samples should be assayed either in real time by laboratories undergoing quality assurance or in batch with short-term storage (less than 1 year). (ii) When real-time assays are to be performed, the serum samples should not be stored at 4 degrees C, but should be frozen immediately after processing and stored frozen until tested.
An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis
Authors: Priem S , Rittig M G , Kamradt T , Burmester G R , Krause A
Published: January 2020

The present study aimed at developing an optimized PCR protocol fro the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCR-positive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.
Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level
Authors: Telenti A , Honoré N , Bernasconi C , March J , Ortega A , Heym B , Takiff H E , Cole S T
Published: January 2020

Progress in understanding the basis of resistance to isoniazid (INH) and rifampin (RMP) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. Consecutive isolates (n = 95) of Mycobacterium tuberculosis, from a Spanish reference laboratory investigating outbreaks of multidrug-resistant tuberculosis, were coded and sent to two external laboratories for genotypic analysis of INH and RMP resistance by PCR-single-strand conformation polymorphism (SSCP) analysis of specific regions of four genes: part of the coding sequence of katG and the promoter regions of inhA and ahpC for INH and the RMP resistance region of rpoB. After correction for the presence of outbreak strains and multiple isolates from single patients, RMP resistance was detected successfully by PCR-SSCP in > 96% of the RMP-resistant strains. PCR-SSCP had a sensitivity of 87% for INH resistance detection, and mutations in katG, inhA, katG-inhA, ahpC, and katG-ahpC were identified in 36.8, 31.6, 2.6, 13.2, and 2.6%, respectively, of the unique strains. Specificity was 100%. Molecular detection of resistance to the two main antituberculous drugs, INH and RMP, can be accomplished accurately by using a strategy which limits analysis to four genetic regions. This may allow the expedient analysis of drug resistance by reference laboratories.
Stability of hybridization activity of Coccidioides immitis in live and heat-killed frozen cultures tested by AccuProbe Coccidioides immitis culture identification test
Authors: Valesco M , Johnston K
Published: January 2020

Frozen hyphal suspensions of Coccidioides immitis were evaluated for suitability as positive control cultures in the AccuProbe C. immitis culture identification test. The genetic probe hybridization activity of heat-killed and viable frozen cultures, stored at -20 and -70 degrees C and tested over a 10-month period, was compared to that of a freshly grown culture, and the results were evaluated based upon the manufacturer's established positive and negative photometric light unit (PLU) cutoff values. All C. immitis suspensions produced positive hybridization values well above the positive and negative cutoff values, and no significant decrease in hybridization activity was observed with the frozen cultures after 10 months of storage. The frozen, heat-killed suspensions produced PLU values with less variability (coefficient of variation, 8% over 10 months than the fresh or frozen viable cultures and were deemed the most stable and sale form of positive control material to use.
Reproducibility of quantitative cultures of endotracheal aspirates from mechanically ventilated patients
Authors: Bergmans D C , Bonten M J , De Leeuw P W , Stobberingh E E
Published: January 2020

Ventilator-associated pneumonia is frequently diagnosed with quantitative cultures of samples obtained by bronchoscopic techniques, a method associated with high costs and potential adverse effects. Quantitative cultures of endotracheal aspirates are easier and cheaper to obtain, and good correlations between the results of this method and those of bronchoscopic methods have been reported. However, the reproducibility of quantitative cultures of endotracheal aspirates has never been determined. We studied the quantitative analysis of endotracheal aspirates from 21 mechanically ventilated patients taken during two study days with 2- and 6-h intervals between samplings. In all, 40 endotracheal aspirates were obtained. For mechanically ventilated patients, the median variation of quantitative culture results was 12.3% (range, 0 to 63%), corresponding to 0.7 log CFU/ml. Furthermore, variation was independent of the interval of time between samplings. Persistence of significant numbers of pathogens in quantitative cultures (> or = 10(5) CFU/ml) of the consecutive endotracheal aspirates occurred in 82% of samples. We conclude that results of quantitative cultures from endotracheal aspirates are reproducible and may be useful in diagnosing ventilator-associated pneumonia.
Molecular fingerprinting of Mycobacterium tuberculosis on a Caribbean island with IS6110 and DRr probes
Authors: Sola C , Horgen L , Goh K S , Rastogi N
Published: January 2020

Because of a substantial increase in human immunodeficiency virus (HIV) infection and HIV-linked tuberculosis in the Caribbean, a molecular fingerprinting study of clinical isolates of Mycobacterium tuberculosis isolated at the Pasteur Institute of Guadeloupe from 1994 to 1995 was undertaken with the insertion sequence IS6110 and the direct repeat DRr probes. We present the results for 72 isolates from 51 patients. A major cluster (cluster A) representing isolates from 12 patients (24%) was detected upon PvuII-IS6110 fingerprinting, which revealed a pattern of four bands among these isolates. Homogeneity was retained when the isolates were further analyzed with the DRr probe or further characterized by AluI and SmaI-DRr restriction fragment length polymorphism analysis. The isolates of cluster A, from 10 men and 2 women, was present in people of all ages and of different ethnic and geographical backgrounds, and infection with these isolates was independent of the HIV status of the patients (except for 2 HIV-positive patients from the same ward from whom the tubercle bacilli were isolated at the same time). The percentage of reactivation versus active transmission events could not be precisely determined in this study. These results are discussed on the basis of the genetic advantage of predominant clusters and/or specific characteristics of the settings from which a similar cluster of isolates with four bands has so far been reported, which include South Africa, French Polynesia, and Guadeloupe.
Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones
Authors: Deguchi T , Yasuda M , Nakano M , Kanematsu E , Ozeki S , Nishino Y , Ezaki T , Maeda S , Saito I , Kawada Y
Published: January 2020

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.
Rapid screening method for identification of cholera toxin-producing Vibrio cholerae O1 and O139
Authors: Osawa R , Okitsu T , Sata S , Yamai S
Published: January 2020

A novel method of identifying cholera enterotoxin (CT)-producing Vibrio cholerae serogroups O1 and O139 was developed. The method uses degradation of NAD as a specific biochemical marker for the CT-producing strains. The substrate NAD at a concentration of 100 mumol/liter was markedly degraded when it was incubated at 37 degrees C for 2 h with the CT-producing stains at a final cell density equivalent to that of a twofold dilution of a McFarland no. 1 standard. NAD degradation was monitored by an enzyme-amplified color development assay. Subsequent tests conducted with a total of 119 strains of V. cholerae, including both clinical and environmental isolates, confirmed a significant correlation between NAD degradation and CT production for all V. cholerae strains belonging to serogroups O1 and O139. Since 2 of 11 non-O1, non-O139 V. cholerae strains not carrying the CT gene degraded NAD, serotyping of the strains prior to the test is recommended.
Ability of commercial ligase chain reaction and PCR assays to diagnose Chlamydia trachomatis infections in men by testing first-void urine
Authors: Chernesky M A , Chong S , Jang D , Luinstra K , Sellors J , Mahony J B
Published: January 2020

A total of 287 men (37.6% with symptoms of urethritis) attending a hospital-based sexually transmitted disease clinic had urethral swabs tested by culture and by direct fluorescent-antibody assay. First-void urine (FVU) was tested for Chlamydia trachomatis by commercially available ligase chain reaction (LCR) and PCR assays. By using an expanded reference standard, 35 men (12.2%) were found to be positive. By performing LCR and PCR, the infection prevalence was found to be approximately twice (11.5 and 12.2%, respectively) that determined swab testing. The sensitivity values were 94.3% for LCR and 100% for PCR. One of the two positive specimens missed by LCR contained inhibitors. PCR produced five false-positive results and LCR produced one.
Molecular cloning and characterization of a recombinant Histoplasma capsulatum antigen for antibody-based diagnosis of human histoplasmosis
Authors: Chandrashekar R , Curtis K C , Rawot B W , Kobayashi G S , Weil G J
Published: January 2020

Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.
Comparison of the BacT/Alert FAN aerobic and the Difco ESP 80A aerobic bottles for pediatric blood cultures
Authors: Welby-Sellenriek P L , Keller D S , Ferrett R J , Storch G A
Published: January 2020

We compared the BacT/Alert system using the aerobic FAN bottle with the ESP system using the 80A aerobic bottle for the detection of pediatric bloodstream pathogens at a children's hospital. From 6,636 blood culture sets complying with the inclusion criteria, 308 pathogens were detected, including 177 that were detected by both systems, 69 that were detected by BacT/Alert FAN only, and 62 that were detected by ESP 80A only (P = 0.6; not significant). BacT/Alert FAN detected more isolates of Staphylococcus aureus (47 versus 34; P = 0.02), while ESP 80A detected more episodes of streptococcal and enterococcal infection. BacT/Alert FAN detected more pathogens from patients receiving antibiotic therapy (107 versus 93; P = 0.04). Of 248 separate episodes of bacteremia or fungemia, 146 were detected by both systems, 56 were detected by ESP 80A only, and 46 were detected by BacT/Alert FAN only (P = 0.37; not significant). The median times to detection were 13.6 h for ESP 80A and 15.7 h for BacT/Alert FAN (P < 0.001). Both systems were considered easy to operate and were free from significant mechanical difficulties. False-positive or false-negative signals were rare or nonexistent with both systems. We conclude that both systems rapidly detect a broad range of pediatric bloodstream pathogens. BacT/Alert FAN provides better detection of Staphylococcus aureus, especially from patients receiving antibiotics. ESP 80A provides better detection of streptococci and enterococci.
Rapid identification and fingerprinting of Candida krusei by PCR-based amplification of the species-specific repetitive polymorphic sequence CKRS-1
Authors: Carlotti A , Chaib F , Couble A , Bourgeois N , Blanchard V , Villard J
Published: January 2020

A PCR method was developed to identify and fingerprint Candida krusei isolates simply and rapidly. The primer pair Arno1 and Arno2 was designed to amplify the polymorphic species-specific repetitive sequence CKRS-1 (C. krusei repeated sequence 1) that we identified in the nontranscribed intergenic regions (IGRs) of rRNA genes in C. krusei LMCK31. The specificity, sensitivity, reproducibility, and fingerprinting ability of the PCR assay were evaluated. Amplification products were obtained from all 131 C. krusei isolates studied. No other yeast species of medical importance (n = 26), including species similar to C. krusei, species of pathogenic filamentous fungi, or a variety of pathogenic bacteria, yielded a PCR product with these primers. This PCR assay allowed for the identification of C. krusei in less than 6 h. The PCR assay was sensitive enough to detect as little as 10 to 100 fg of C. krusei-purified DNA and proved to be reproducible. Since amplification products varied both in number and in molecular weight according to the strains, PCR patterns allowed strains to be distinguished. To ascertain the epidemiological usefulness of this PCR fingerprinting, the patterns of the 131 isolates were compared. A total of 95 types which corresponded to 95 independent strains were delineated (discriminatory power = 1 with n = 95). Comparison of the results of PCR fingerprinting and those of fingerprinting with the CkF1,2 probe showed that they concurred. In addition, this work yields insights into the mechanisms involved in generating polymorphisms in the IGRs of C. krusei. Since this method is simpler and faster than established identification and genotyping methods of this important pathogenic species, it is a critical improvement for clinical microbiology laboratories relevant not only to diagnosis but also to epidemiology.