Gene Polymerase Chain Reaction Assay Research Articles

Diagnostic Modality of Clostridioides difficile Infection Predicts Treatment Response and Outcomes in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) patients are at increased risk of developing Clostridioides difficile infection (CDI). Common methods to diagnose CDI involve a combination of tests including the toxin A/B enzyme immunoassay (Toxin) and toxin gene polymerase chain reaction assay (PCR). Disease outcomes in Toxin+ versus Toxin-PCR+IBD patients remain unclear. This study aimed to examine the response to antibiotics and risk of IBD therapy escalation in Toxin+ versus Toxin-PCR+ patients. IBD patients at an academic center with CDI diagnosis based on Toxin+ or Toxin-PCR+ from 2012 to 2017 were identified. Comparisons of response to antibiotics within 30days and escalation of IBD therapy within 90days of CDI diagnosis between these two groups were analyzed by Chi-square analysis. Multivariable regression analysis examined factors associated with antibiotic response. Among 92 patients included, 61% had Crohn's diseaseand 39% had ulcerative colitis. 70% tested Toxin-PCR+. 60% received vancomycin or fidaxomicin to treat CDI. 82% of Toxin+ patients responded to antibiotics compared to 25% of Toxin-PCR+ patients (p < 0.001). 21% of Toxin+ patients required IBD therapy escalation compared to 63% of Toxin-PCR+ patients (p < 0.001). When adjusted for the types of antibiotics used, IBD subtypes, and immunosuppression status, positivity to Toxin (OR 14.85, CI 4.62-47.72) was the most significant predictor of response to antibiotics. Toxin+ compared to Toxin-PCR+IBD patients had a significantly higher rate of response to antibiotics and lower chances of requiring IBD therapy escalation. Future outcome studies involving CDI in IBD patients should be stratified by modality of diagnosis.

2358. Understanding the Clinical Implications of Clostridium difficile Detection in the Molecular Age: Colonization vs. Infection in Children Less Than 3 Years of Age

BackgroundInfants have a high rate of asymptomatic Clostridium difficile (CD) colonization (up to 37%) but can rarely develop true CD infection (CDI). However, currently available polymerase chain reaction (PCR) and enzyme immunoassays (EIA) have suboptimal sensitivity/specificity to distinguish CDI from colonization. Recent data from adults showed that lower cycle threshold (Ct) values of a semi-quantitative CD toxin B gene (tcdB) PCR assay in stool correlated with detection of free CD toxin in stool and poor clinical outcomes. We hypothesized that a tcdB PCR assay may be utilized to distinguish CDI from colonization in patients < 3 years old.MethodsSymptomatic patients < 3 years old with CD detected by the BioFire FilmArray Gastrointestinal Panel (FGP) were enrolled 2/2018–3/2019. We performed CD tcdB PCR and toxin A/B/GDH EIA on frozen aliquots of stool in Cary Blair. CDI was defined among those that were tcdB PCR positive as (1) a consistent clinical syndrome (diarrhea + no current laxative use), (2) CD EIA toxin+, (3) symptomatic improvement with CDI-directed treatment, and (4) no alternative etiology of diarrhea identified. Patients who did not meet criteria for CDI were considered colonized. We compared median tcdB PCR Ct values between the CDI and colonized groups using the Mann–Whitney test.ResultsOf 193 FGP CD+ patient samples with charts available for review, 37 (19%) samples were EIA GDH+/toxin+, 121 (63%) were GDH+/toxin− and 35 (18%) were EIA−. 150 (78%) samples had detectable tcdB by PCR. Six (4%) patients met criteria for CDI and 144 (96%) for colonization. Median (interquartile range) tcdB PCR Ct values were 23.8 (22.0–29.5) and 30.5 (26.3–35.8) in patients with CDI and colonization, respectively (P = 0.03).ConclusionUsing a strict clinical and laboratory definition, 4% of evaluable patients < 3 years old met criteria for CDI and had significantly lower tcdB PCR Ct values than colonized patients. A combination of clinical and laboratory criteria, including semi-quantitative tcdB PCR, may help differentiate colonization from CDI in this patient population.Disclosures All authors: No reported disclosures.

Antibiotic Resistance Patterns and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus in Clinical Settings in Rwanda.

The escalating burden of infections attributable to methicillin-resistant Staphylococcus aureus (MRSA) in East African countries is calling for interventional strategies to control the spread of this strain. The present study aimed at determining the prevalence, antimicrobial profiles, and staphylococcal cassette chromosome mec (SCCmec) typing of MRSA strains. This was a cross-sectional laboratory-based study involving 226 non-duplicated S. aureus isolates from different clinical samples of patients attending a referral hospital in Kigali. Kirby-Bauer disk diffusion method was used for drug susceptibility testing. Methicillin-resistant S. aureus were confirmed using polymerase chain reaction (PCR) assay for the mecA gene and SCCmec type PCR assay was used for genotyping. Of 138 S. aureus, 39 (31.2%) were found to be MRSA strains. The mean age of the patients was 21.9 years. The incidence of MRSA increases with age and was 27.1% in patient age group younger than 18 years, 33.3% in the age group between 19 and 65 years, and 66.7% in patient age group older than 65 years. There was a significant association between geographic regions and incidence of MRSA (P = 0.02) with the high MRSA isolates from Northern (61.5%) and Western (50%) provinces. Methicillin-resistant S. aureus strains were found to be mostly susceptible to linezolid (93.5%). Among the MRSA strains, SCCmec type I and SCCmec type IV were the most prevalent at 56.4% and 17.9%, respectively. A high prevalence of MRSA was found in Rwanda. Staphylococcal cassette chromosome mec type I (52.2%) was the most predominant. A continuous surveillance of MRSA strains, particularly in the hospital settings, should be an enduring exercise in Rwanda.

Microbiological and Molecular Investigation of Clinically Suspected Caseous Lymphadenitis Cases in Goats

Chronic suppurative lymphadenitis characterized by abscess formation is of economic significance in sheep and goats. It is principally caused by Corynebacterium pseudotuberculosis (caseous lymphadenitis, CLA) and Staphylococcus aureus subsp. anaerobius. Pus samples from superficial lymph nodes of 33 (4.38 %) out of 752 adult Sirohi goats showing clinical lesions similar to CLA were collected for laboratory investigations. Sixteen (48.48 %) bacterial isolates from pus were identified as C. pseudotuberculosis, 12 (36.36 %) as catalase-negative S. aureus subsp. anaerobius while samples from 5 cases were negative. The polymerase chain reaction (PCR) assays targeting putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate oxidoreductase coenzyme F420-dependent and proline iminopeptidase genes of C. pseudotuberculosis when carried out directly on the DNA extracted from pus were positive in the same 16 goats. All the C. pseudotuberculosis isolates were also found to be positive for these genes in the PCR. Isolates of S. aureus subsp. anaerobius were found to be positive for 16S rRNA and nuclease (nuc) gene PCR. The present study shows the incidence of CLA as 4.38, 2.12 and 2.12 % based on the clinical, bacterial culture and direct PCR assay on pus samples, respectively. The incidence of abscess disease due to S. aureus subsp. anaerobius was 1.59 %. The three gene PCR assay developed in the study was found to be specific and rapid than the bacterial culture in detecting bacteria directly in the pus samples and can be applied for the diagnosis and control strategy of CLA.

Molecular detection of DHFR gene polymorphisms in Pneumocystis jirovecii isolates from Indian patients.

Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India. Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms. Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and 10% (18/180) were positive by mt LSU rRNA PCR assay. Of these 18 positive samples, only 77% (14/18) were amplified by the DHFR gene PCR assay. A total of 16 nucleotide substitutions were observed in 42% (6/14) samples targeted for the DHFR gene, of which 8 nucleotide substitutions were synonymous and the rest were non-synonymous. The DHFR gene mutations found in this study may possibly indicate an association of process likely to contribute to therapeutic failure or an evolutionary process, and warrant continuous monitoring.

Comparison of antigen and two molecular methods for the detection of Clostridium difficile toxins

Clostridium difficile (CD) is considered an important cause of diarrhoea associated with the antimicrobial treatment of infections. The pathogenicity of CD is due to toxins A and B, produced by toxigenic CD strains. We evaluated 3 methods for detecting CD toxins: the RIDASCREEN® enzyme immunoassay (EIA) (R-Biopharm)--one detecting toxins directly in the stool specimens and another detecting toxins from isolated CD strains--and 2 molecular methods, the illumigene™ loop-mediated isothermal amplification (LAMP) assay (Meridian) and RIDA®GENE polymerase chain reaction (PCR) assay (R-Biopharm), as direct identification methods from stool specimens. Toxigenic culture (TC) was used as the reference method. Altogether 884 stool samples were analyzed, of which 253 (29%) were positive by TC. Six hundred and seventy-two specimens were tested by RIDASCREEN EIA, 430 were tested with the illumigene LAMP assay, and 212 were tested with the RIDA GENE PCR assay. CD toxin A and B antigen tests by EIA were very insensitive, both directly from stool specimens (2 series; 57-61%) and in isolated CD strains (53%); consequently the negative predictive value remained low (84-93% and 91%, respectively). Specificity, however, was very good at 98-100%. The 2 molecular methods detected CD toxin genes excellently and equally, resulting in sensitivities, specificities, and positive and negative predictive values of 98%, 100%, 100%, and 98%, respectively. Both molecular assays were easy to use, rapid, sensitive, and specific for the detection of toxigenic CD strains.

Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays

The performance of C. Diff Quik Chek Complete (QCC), BD GeneOhm Cdiff PCR (BD), and ProGastro Cd PCR (PG) assays was evaluated in detecting Clostridium difficile infection (CDI) in children using 200 frozen stool specimens. The results of the tests were compared to the toxigenic culture (TC) as ‘gold standard.’ The sensitivity, specificity, positive predictive value, and negative predictive value were as follows. QCC antigen (GDH + Toxin-A/B) = 70.8%, 97.4%, 89.5%, and 91.4%; BD PCR = 89.6%, 96.7%, 89.6%, and 96.7%; PG PCR = 100%, 93.4%, 82.8%, and 100%. Polymerase chain reaction (PCR) assays detected an additional 11 positives missed by TC, 7 of which were confirmed positive by an alternate tcdB gene PCR assay. However, retrospective clinical chart review indicated CDI in only 3 of the 11 patients in whom C. difficile was detected by PCR only. A 2-step algorithm utilizing QCC antigen test as a screening test followed by confirmation of GDH-positive and toxin-negative samples with either BD or PG PCR assay will provide rapid and accurate results for majority of the samples and reduce laboratory testing cost.

A VP3/VP1 gene polymerase chain reaction assay for detection of chicken anemia virus in broiler samples

A PCR assay was designed for amplification of the highly conserved VP3 gene and a 5' region of the VP1 gene, for the diagnosis of CAV in organ samples of broiler flocks suspected of chicken infectious anemia. A comparison of the VP3/VP1 PCR with in vivo virus isolation revealed 100% agreement of the results, with 13 positive and 3 negative samples in both assays, indicating that the VP3/VP1 PCR is a specific diagnostic method. Tissues from additional 24 broiler chicken flocks, with CAV-like lesions and clinical history were then tested only by the VP3/VP1 PCR and a reference PCR with published primers for the VP1 gene. Nineteen samples resulted positive and one negative in both PCR, while another 4 samples were positive only in the VP3/VP1 PCR. These results indicate that the VP3/VP1 PCR is a sensitive, specific diagnostic test, suitable as an alternative to the expensive and time consuming in vivo virus isolation method, specially considering the difficult diagnosis of CAV strains not readily adaptable to MSB-1 cell culture.

B-Cell Clonality Determination Using an Immunoglobulin κ Light Chain Polymerase Chain Reaction Method

To augment the detection of clonality in B-cell malignancies, we designed a consensus primer kappa light chain gene (Igkappa) polymerase chain reaction (PCR) assay in combination with a consensus primer immunoglobulin heavy chain gene (IgH) PCR assay. Its efficacy was then evaluated in a series of 86 paraffin tissue samples comprising neoplastic and reactive lymphoproliferations. Analysis after PCR was accomplished by 10% native polyacrylamide gel electrophoresis after heteroduplex pretreatment of PCR products and by a post-PCR chip-based capillary electrophoresis analytic method. Overall, 49 of 68 (72%) of mature B-cell neoplasms yielded discrete Igkappa gel bands within the predicted size range with no clonotypic Igkappa products observed among reactive lymphoid or T-cell proliferations. The application of Igkappa PCR improved overall sensitivity from 81% with IgH PCR alone to 90% with combined Igkappa/IgH PCR, with this effect being most notable in germinal center-related lymphomas. Sequencing of positive Igkappa rearrangements revealed that most rearrangements involved members of the Vkappa1 (40%) and Vkappa2 (34%) gene families along with Jkappa1 (26%), Jkappa2 (23%), and Jkappa4 (51%) gene segments. Involvement of Vkappa pseudogenes was identified in 24% of cases with Vkappa-KDE rearrangements. Our results demonstrate the efficacy of Igkappa PCR in improving the detection rate of clonality in B-cell neoplasms and further introduce a novel post-PCR chip-based capillary electrophoresis analytic method for rapid PCR fragment size evaluation.

Prevalence of the Enterotoxin Gene and Clonality of Clostridium perfringens Strains Associated with Food-Poisoning Outbreaks

The prevalence of the enterotoxin gene in a well-characterized collection of 71 Clostridium perfringens strains from 36 separate food-poisoning cases or outbreaks was analyzed with the polymerase chain reaction (PCR). The clonality of 39 strains originating from 14 outbreaks where at least two isolates were available was studied with pulsed-field gel electrophoresis (PFGE) using SmaI and ApaI restriction endonucleases. The cpe gene PCR assay was found to correlate well with Clostridium perfringens enterotoxin (CPE) production in vitro with reverse passive latex agglutination. Of the C. perfringens food and clinical food-poisoning isolates 24 (86%) and 38 (88%) were cpe-positive, respectively. Different PFGE patterns indicated that multiple cpe-positive clones are frequently present within one outbreak. The existence of cpe-positive and negative isolates with identical or nearly identical PFGE patterns in a single outbreak suggests that the cpe gene may be in a movable genetic element.

Phenotype, genotype and clonality of Reed-Sternberg cells in nodular sclerosis Hodgkin's disease: results of a single-cell study.

The genotype and clonality of Reed-Sternberg (RS) cells in Hodgkin's disease (HD) has remained a controversial issue, largely due to the scarcity of RS cells in tissues and the limitations of the techniques used to resolve this issue. Southern hybridization and polymerase chain reaction (PCR) assays using DNA extracted from tissues can only document clonal gene rearrangements, but do not indicate which cellular population is responsible for such rearrangements. To overcome the limitations of these previous techniques for studying the genotype and clonality of RS cells, we analysed single RS cells with a single-cell PCR assay to detect immunoglobulin heavy chain gene (IgH) rearrangements and X-chromosome inactivation. Six cases of nodular sclerosis (NS) HD were studied. The RS cells displayed a B-cell phenotype in three cases and a null-cell phenotype in the other three cases. IgH rearrangements were detected in the RS cells of the three cases with a B-cell phenotype, but not in the other cases. In these three cases the IgH rearrangements in the RS cells were polyclonal, although a subpopulation of clonal RS cells was documented in one case. The finding that the RS cells with IgH rearrangements were not monoclonal was supported in one case by studying the pattern of X-chromosome inactivation in single RS cells by a single-cell human androgen receptor gene (HUMARA) PCR assay. Our results indicate that NSHD begins as a polyclonal process in which a clonal RS cell population may arise; and that the RS cells in a subset of NSHD show evidence of B-lineage differentiation.