Economic appraisal of using genetics to control Streptococcus agalactiae in Nile tilapia under cage and pond farming system in Malaysia.

Disease outbreaks have been seen as the major threat to sustainable aquaculture worldwide. Injectable vaccines have been one of the few strategies available to control the diseases, however, the adoption of this technology globally is limited. Genetic selection for disease resistance has been proposed as the alternative strategy in livestock and aquaculture. Economic analysis for such strategies is lacking and this study assesses the economic worth of using tilapia fingerlings resistant to Streptococcosis in both cage and pond production systems. The paper also assesses the profitability of paying the higher price for such fingerlings. Partial-budgeting was used to develop a stochastic simulation model that considers the benefits and costs associated with the adoption of tilapia fingerlings resistant to Streptococcosis at the farm level, in one production cycle. In both ponds and cage production systems, the use of genetically selected Streptococcus resistant tilapia fingerlings was found to be profitable where Streptococcus infection is prevalent. In the cages and ponds where Streptococcus related mortality was ≥ 10%, the Nile tilapia aquaculture was found to be profitable even if the amount paid for genetically selected Streptococcus resistant tilapia fingerlings was 100% higher than the amount paid for standard fingerlings.

Genome-Wide Assessment of Streptococcus agalactiae Genes Required for Survival in Human Whole Blood and Plasma.

Streptococcus agalactiae (group B streptococcus, or GBS) is a common cause of bacteremia and sepsis in newborns, pregnant women, and immunocompromised patients. The molecular mechanisms used by GBS to survive and proliferate in blood are not well understood. Here, using a highly virulent GBS strain and transposon-directed insertion site sequencing (TraDIS), we performed genome-wide screens to discover novel GBS genes required for bacterial survival in human whole blood and plasma. The screen identified 85 and 41 genes that are required for GBS growth in whole blood and plasma, respectively. A common set of 29 genes was required in both whole blood and plasma. Targeted gene deletion confirmed that (i) genes encoding methionine transporter (metP) and manganese transporter (mtsA) are crucial for GBS survival in whole blood and plasma, (ii) gene W903_1820, encoding a small multidrug export family protein, contributes significantly to GBS survival in whole blood, (iii) the shikimate pathway gene aroA is essential for GBS growth in whole blood and plasma, and (iv) deletion of srr1, encoding a fibrinogen-binding adhesin, increases GBS survival in whole blood. Our findings provide new insight into the GBS-host interactions in human blood.

Comparative study of Revogene GBS LB assay and GeneXpert GBS LB assay for the detection of group B Streptococcus in prenatal screening samples.

BACKGROUND: Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC therefore recommends GBS screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. METHOD: In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, Revogene GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsin's clinical microbiology laboratory for GBS testing. RESULTS: We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with a positive percent agreement of 98.0% and a negative percent agreement of 96.5% between the Revogene GBS LB assay and the GeneXpert GBS LB assay. CONCLUSION: Overall, we report that both assays perform well for the detection of GBS colonization in pregnant women.

Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women.

Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection.

Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women

ABSTRACT Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection.

Comparison of qPCR and culture methods for group B Streptococcus colonization detection in pregnant women: evaluation of a new qPCR assay.

BACKGROUND: Streptococcus Group B (GBS) colonization in pregnant women is the most important risk factor for newborn disease due to vertical transmission during delivery. GBS colonization during pregnancy has been implicated as a leading cause of perinatal infections. Traditionally, pregnant women are screened for GBS between 35 and 37 weeks of gestation. However, antenatal culture-based screening yields no information on GBS colonization status and offers low predictive value for GBS colonization at delivery. Numerous assays have been evaluated for GBS screening in an attempt to validate a fast and efficient method. The aim of this study was to compare bacteria isolation by culture and two qPCR techniques, targeting sip and cfb genes, respectively, for detecting colonizing GBS. METHODS: Cultures - the gold-standard technique, a previous qPCR technique targeting the sip gene, and a new proposed qPCR assay targeting the cfb gene were evaluated as diagnostic tools on 320 samples. RESULTS: Considering cultures as the gold standard, the evaluated qPCR method detected 75 out of 78 samples, representing a sensitivity of 93.58% (95% confidence interval (CI), 90.89-96.27) and specificity of 94.62% (95% CI, 91.78-97.46). However, an additional analysis was performed for true positives that included not only samples showing positives by culture but samples showing positive for both qPCR assays. The sensitivity and specificity were recalculated including these discrepant samples and a total of 89 samples were considered as positive, giving a prevalence of 27.81%. With this new analysis, the qPCR targeting the cfb gene showed a sensitivity of 95.5% (95% CI, 88.65-98.59) and specificity of 99.13% (95% CI, 96.69-99.97). CONCLUSIONS: The new qPCR method is a sensitive and specific assay for detecting GBS colonization and represents a valuable tool for identifying candidates for intrapartum antibiotic prophylaxis. Cultures should be retained as the reference and the routine technique because of its specificity and cost analysis ratio, but it would be convenient to introduce PCR techniques to check negative culture samples or when an urgent detection is required to reduce risk of infection among infants.

Intrapartum PCR assay versus antepartum culture for assessment of vaginal carriage of group B streptococci in a Danish cohort at birth.

The aim of this study was to compare the performances of two strategies for predicting intrapartum vaginal carriage of group B streptococci (GBS). One strategy was based on an antepartum culture and the other on an intrapartum polymerase chain reaction (PCR). We conducted a prospective observational study enrolling 902 pregnant women offered GBS screening before delivery by two strategies. The Culture-strategy was based on vaginal and rectal cultures at 35-37 weeks' gestation, whereas the PCR-strategy was based on PCR assay on intrapartum vaginal swab samples. An intrapartum vaginal culture for GBS was used as the reference standard from which the performances of the 2 strategies were evaluated. The reference standard showed a GBS-prevalence of 12%. The culture-strategy performed with a sensitivity of 82%, specificity of 91%, positive predictive value (PPV) of 55%, negative predictive value (NPV) of 98%, and Likelihood ratio (LH+) of 9.2. The PCR-strategy showed corresponding values as sensitivity of 83%, specificity of 97%, PPV of 78%, NPV of 98%, and LH+ of 27.5. We conclude that in a Danish population with a low rate of early-onset neonatal infection with GBS, the intrapartum PCR assay performs better than the antepartum culture for identification of GBS vaginal carriers during labor.

Accuracy of a rapid real-time polymerase chain reaction assay for diagnosis of group B Streptococcus colonization in a cohort of HIV-infected pregnant women.

OBJECTIVE: There are limited data regarding Xpert performance to detect Group B Streptococcus (GBS) in HIV-infected pregnant women. We evaluated the accuracy of a rapid real-time polymerase chain reaction (PCR) test in a cohort of HIV-infected women. METHODS: At 35-37 weeks of pregnancy, a pair of combined rectovaginal swabs were collected for two GBS assays in a cohort of sequentially included HIV-infected women in Rio de Janeiro: (1) culture; and (2) real-time PCR assay [GeneXpert GBS (Cepheid, Sunnyvale, CA)]. Using culture as the reference, sensitivity, specificity, positive and negative-likelihood ratios were estimated. RESULTS: From June 2012 to February 2015, 337 pregnant women met inclusion criteria. One woman was later excluded, due to failure to obtain a result in the index test; 336 were included in the analyses. The GBS colonization rate was 19.04%. Sensitivity and specificity of the GeneXpert GBS assay were 85.94% (95% CI: 75.38-92.42) and 94.85% (95% CI: 91.55-96.91), respectively. Positive and negative predictive values were 79.71% (95% CI: 68.78-87.51) and 96.63% (95% CI: 93.72-98.22), respectively. CONCLUSIONS: GeneXpert GBS is an acceptable test for the identification of GBS colonization in HIV-infected pregnant women and represents a reasonable option to detect GBS colonization in settings where culture is not feasible.

A new mass spectrometry based bioassay for the direct assessment of hyaluronidase activity and inhibition.

The development of drug resistance by bacterial pathogens is a growing threat. Drug resistant infections have high morbidity and mortality rates, and treatment of these infections is a major burden on the health care system. One potential strategy to prevent the development of drug resistance would be the application of therapeutic strategies that target bacterial virulence. Hyaluronidase is virulence factor that plays a role in the ability of Gram-positive bacteria such as Staphyloccus aureus and Streptococcus agalactiae to spread in tissue. As such, this enzyme could be a target for the development of future anti-virulence therapies. To facilitate the identification of hyaluronidase inhibitors, quantitative and reproducible assays of hyaluronidase activity are required. In the present study, we developed a new mass spectrometry based bioassay for this purpose. This assay directly measures the quantity of a degradation product (3-(4-deoxy-ß-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine) produced by the hyaluronidase enzyme. Validation parameters for the new assay are as follows: repeatability, <7%; intermediate precision, <10%; range, 0.78-50 µM; limit of detection, 0.29 µM; and limit of quantification, 0.78 µM. Using the new assay, the IC50 value for a published inhibitor of S. agalactiae hyaluronidase, ascorbic acyl 6-palmitate, was 8.0±1.0 µM. We also identified a new hyaluronidase inhibitor, n-cyclohexanecarbonylpentadecylamine, with an IC50 of 30.4±9.8 µM. In conclusion, we describe a new, direct, and reproducible method for assessing hyaluronidase activity using mass spectrometry that can facilitate the discovery of inhibitors.

A multi locus variable number of tandem repeat analysis (MLVA) scheme for Streptococcus agalactiae genotyping.

BACKGROUND: Multilocus sequence typing (MLST) is currently the reference method for genotyping Streptococcus agalactiae strains, the leading cause of infectious disease in newborns and a major cause of disease in immunocompromised children and adults. We describe here a genotyping method based on multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) applied to a population of S. agalactiae strains of various origins characterized by MLST and serotyping. RESULTS: We studied a collection of 186 strains isolated from humans and cattle and three reference strains (A909, NEM316 and 2603 V/R). Among 34 VNTRs, 6 polymorphic VNTRs loci were selected for use in genotyping of the bacterial population. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each VNTR locus. 98 MLVA genotypes were obtained compared to 51 sequences types generated by MLST. The MLVA scheme generated clusters which corresponded well to the main clonal complexes obtained by MLST. However it provided a higher discriminatory power. The diversity index obtained with MLVA was 0.960 compared to 0.881 with MLST for this population of strains. CONCLUSIONS: The MLVA scheme proposed here is a rapid, cheap and easy genotyping method generating results suitable for exchange and comparison between different laboratories and for the epidemiologic surveillance of S. agalactiae and analyses of outbreaks.

Assessment of an extraction protocol to detect the major mastitis-causing pathogens in bovine milk.

Despite all efforts to control its spread, mastitis remains the most costly disease for dairy farmers worldwide. One key component of better control of this disease is identification of the causative bacterial agent during udder infections in cows. Mastitis is complex, however, given the diversity of pathogens that must be identified. Development of a rapid and efficient bacterial species identification tool is thus necessary. This study was conducted to demonstrate the feasibility of bacterial DNA extraction for the automated molecular detection of major mastitis-causing pathogens directly in milk samples to complement traditional microbiological identification. Extraction and detection procedures were designed and optimized to achieve detection in a respectable time frame, at a reasonable cost, and with a high throughput capacity. The following species were identified: Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Klebsiella spp. (including Klebsiella oxytoca and Klebsiella pneumoniae). The detection procedure includes specific genomic DNA amplification by multiplex PCR for each species, separation by capillary electrophoresis, and laser-assisted automated detection. The specificity of the primers was assessed with a panel of bacteria representing mastitis-negative control species. The extraction protocol comprised multiple steps, starting with centrifugation for fat removal, followed by heating in the presence of a cation exchange resin to trap divalent ions. The analytical sensitivity was 100 cfu/mL for milk samples spiked with Staph. aureus, Strep. dysgalactiae, and E. coli, with a tendency for K. pneumoniae. The detection limit was 500 cfu/mL for Strep. uberis and Strep. agalactiae. The overall diagnostic sensitivity (95.4%) and specificity (97.3%) were determined in a double-blind randomized assay by processing 172 clinical milk samples with microbiological characterization as the gold standard. When the physical nature of the milk samples was too altered, DNA purification with a magnetic bead-based system was used. Of the apparent false-positive samples, 5 were identified by specific microbiological analysis as true-positive Staph. aureus co-infections, with further confirmation by ribosomal 16S sequencing. The proposed methodology could, therefore, become an interesting tool for automated PCR detection of major mastitis pathogens in dairy cattle.

Real-time PCR assay provides reliable assessment of intrapartum carriage of group B Streptococcus.

The objective of this study was to determine the reliability of the real-time PCR assay for determining the group B Streptococcus (GBS) status of women in labor. In this prospective study we compared the results of culture and PCR testing of vaginal and rectal samples collected by nursing staff when women were in labor. Patients' charts were also reviewed to obtain relevant information about pregnancy risk factors. Our results demonstrated a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 90.5%, 96.1%, 86.4%, and 97.4%, respectively, for rapid PCR. Of the 196 women evaluated, 29 (14.8%) presented with unknown GBS status, 11 (37.9%) of whom received unnecessary intrapartum antibiotics. The rapid real-time PCR test was robust and was able to reliably detect the presence of GBS in women in labor within 1 h of specimen submission to the laboratory. We recommend that the rapid PCR test be targeted to women who present in labor with unknown GBS status. In cases where the laboratory does not offer 24-h availability of testing, sample collection followed by PCR testing the next morning is still valuable and provides reliable results 24 to 48 h faster than culture and will aid appropriate decision-making regarding continuing or stopping antibiotics for neonates of women with unknown GBS status.

The putative glycosyltransferase-encoding gene cylJ and the group B Streptococcus (GBS)-specific gene cylK modulate hemolysin production and virulence of GBS.

Group B streptococcus (GBS) expresses a hemolysin/cytolysin that plays an important role in pathogenesis. Using the Himar1 transposon mutagenesis system, a hypohemolytic mutant carrying an interrupted cylJ gene was characterized. cylJ, encoding a putative glycosyltransferase, and cylK, whose product is unknown, are both required for the full hemolytic/cytolytic activity, pigment formation, and virulence of GBS.

Comparison of LightCycler PCR and culture for detection of group B streptococci from vaginal swabs.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. New rapid, sensitive and specific methods for detection of GBS in pregnant women are needed in order to provide timely treatment of neonates. The sensitivity, specificity and cost of a LightCycler PCR method was compared with selective culture for the detection of GBS from 400 vaginal swabs. In addition, two DNA extraction methods (simple boiling and automated DNA extraction by Roche MagNA Pure LC) were compared for a subgroup of 100 clinical samples. The sensitivity of the LightCycler PCR assay for the detection of GBS from vaginal swabs was significantly higher than that of culture. There were no culture-positive, LightCycler PCR-negative cases. The efficiencies of the two DNA extraction procedures were not significantly different. The detection of GBS from vaginal swabs by the molecular method (including simple boiling extraction) required the same hands-on time, but the procedure was completed in 1.5 h, compared with c. 48 h for the culture-based approach. Disadvantages of the molecular method are the increased costs (45%) and the absence of antibiogram data. The LightCycler PCR is a promising tool for sensitive, specific and rapid detection of GBS directly from clinical specimens of pregnant women.

Rapid detection of group B streptococcus and Escherichia coli in amniotic fluid using real-time fluorescent PCR.

OBJECTIVE: To establish reliability and validity of real-time fluorescent PCR for early detection of bacterial invasion of the amniotic cavity. METHODS: Amniotic fluid samples from 40 patients undergoing mid-trimester genetic amniocentesis were incubated for 6 h at 37 degrees C and were cultured on media specific for group B streptococcus (GBS) and E. coli. Concurrently, samples were analyzed with real-time fluorescent PCR (Roche LightCycler) using DNA primers and probes designed to detect the CAMP factor encoding cfb gene and uidA gene of GBS and E. coli, respectively. For positive control and to simulate amniotic fluid colonization, 104 cfu/ml of GBS and E. coli were inoculated on sterile amniotic fluid and incubated for 6 h. Bacterial genomic DNA for the two organisms was extracted and purified via the two-step precipitation method using a commercial kit. The real-time PCR assays were also tested against 25 non-GBS and non-E. coli bacterial species. The lower limit of detection for each pathogen was established using serial dilution of bacterial genomic DNA. RESULTS: All patient samples were negative for evidence of GBS and E. coli with both culture and real-time PCR methods. Amniotic fluid samples inoculated with GBS and E. coli were positive with real-time PCR whereas the 25 bacterial species other than GBS or E. coli tested negative with the assay. Average total sample processing time including the pre-enrichment step was 7 h 40 min. The average cost for DNA extraction and PCR testing was 8.50 dollars per test. CONCLUSION: Real-time fluorescent PCR is a valid and reliable method for detection of specific pathogens in amniotic fluid. This technique is sensitive for low inoculation levels. Real-time fluorescent PCR has potential to impact clinical management as a rapid, reliable detection method for GBS and E. coli in chorioamnionitis.

Genetic heterogeneity of the pathogenic potentials of human and bovine group B streptococci.

One-hundred seventy-two B-streptococcal strains of human and bovine origin were analyzed for the presence of 9 genes potentially involved in virulence. Some of genes (glnA, cyl, hylB, scaA and cfb) were revealed in all the strains. However, the presence of others (bca, bac, scpB, lmb) varied from strain to strain. Taken together, 3 and 5 different types of pathogenic potential were found among human and bovine group B streptococci (GBS) strains, respectively, and only one type (bca+ bac scpB+ glnA+ cyl+ hylB+ lmb+ scaA+ cfb+) was common for both kinds of strains. We propose that different virulence genes can be involved in the development of infectious processes in humans and animals. A reliable PCR protocol with 3 pairs of primers (for the genes bca, bac and scpB) in the same reaction mixture was developed for the fast identification of the pathogenic potential of GBS. In comparison with the classical immunological methods this procedure displayed higher specificity and sensitivity as well as a shorter time of analysis. It can be recommended for use in the clinical and veterinary practice for studying the epidemiological relationship between the isolates and the ready identification of the clone causing the infection.