Qualidade do leite cru refrigerado avaliado por diferentes laboratórios credenciados pelo MAPA / Quality of refrigerated raw milk evaluated by different laboratories accredited by MAPA

O objetivo deste trabalho foi analisar e correlacionar os resultados de qualidade do leite cru refrigerado de dez propriedades rurais de Viçosa (MG) obtidos por métodos de referências do Ministério da Agricultura, Pecuária e Abastecimento e por espectrofotômetros de luz infravermelha em três laboratórios (A, B e C) credenciados pelo mesmo órgão de fiscalização. As amostras de leite foram analisadas quanto à contagem bacteriana, contagem de células somáticas (CCS) e teores de gordura, proteína e extrato seco desengordurado (ESD). Não houve discordância de amostras de leite quanto aos teores de proteína e gordura. Porcentagens diferentes de discordância foram observadas entre os resultados dos laboratórios e método de referência quanto à CCS, contagem bacteriana e teores de ESD. Os teores médios de proteína dos laboratórios A e B e os teores médios de ESD de todos laboratórios não foram estatisticamente iguais (p<0,05) aos teores obtidos pelo método de referência. Os teores de gordura obtidos no laboratório B não tiveram correlação estatística (p>0,05) com os teores obtidos pelo método de referência. Observou-se que as CCS e contagens bacterianas médias de todos os laboratórios foram estatisticamente iguais (p>0,05) e correlacionadas (p<0,05) às contagens médias obtidas pelos métodos de referência. Conclui-se que os teores de proteína em dois laboratórios e ESD em todos os laboratórios não são equivalentes com os métodos de referência do MAPA. É importante que os laboratórios revejam a calibração dos seus equipamentos quanto à quantificação dos teores de sólidos do leite.

The objective of this study was to analyze and to correlate the results of refrigerated raw milk quality in ten farms of Viçosa (MG). The milk was analyzed by the Ministério da Agricultura, Pecuária e Abastecimento standard methods and by infrared spectrophotometers in three laboratories (A, B and C) accredited by the same institution. The milk samples were analyzed for bacterial counts, somatic cell count (SCC) and fat, protein and solids not fat (SNF) tenors. There were no disagreement milk samples as the protein and fat tenors. Different percentages of disagreement were observed between the results of laboratory and standard method as the SCC, bacterial count and SNF tenor. The protein mean tenor of A and B laboratories and the SNF mean tenor of all laboratories were not statistically equal (p<0.05) the tenors obtained by standard method. The fat tenors obtained in B laboratory were not statistically correlated (p>0.05) with tenors obtained by the standard method. It was observed that the means of SCC and bacterial counts of all laboratories were statistically the same (p>0.05) and correlated (p<0.05) to the means counts obtained by standard methods. It is concluded that the protein tenors in two laboratories and SNF in all laboratories are not equivalent with the reference methods of MAPA. It is important that laboratories review the calibration of the equipment on the quantification of milk solids tenors.

Evaluation of the Genmark ePlex® and QIAstat-Dx® respiratory pathogen panels in detecting bacterial targets in lower respiratory tract specimens.

BACKGROUND: The ePlex® and QIAstat-Dx® respiratory pathogen panels detect multiple respiratory pathogens, mainly viruses but also Legionella pneumophila, Mycoplasma pneumoniae and Bordetella pertussis. The assays have been marketed for use in nasopharyngeal swab specimens. For diagnosing bacterial pneumonia, lower respiratory tract (LRT) specimens are indicated. Aim of this study was to evaluate the performance of these syndromic panels for these three bacterial targets in samples from the LRT. Fifty-six specimens were collected from our repositories, five negative samples and fifty-one samples which had been previously tested positive with the routine diagnostic real-time PCR assays for Legionella spp. (N = 20), Bordetella spp. (N = 16) or M. pneumoniae (N = 15). RESULTS: The QIAstat-Dx Respiratory Panel V2 (RP) assay detected all of the L. pneumophila and B. pertussis positive samples but only 11/15 (73.3 %) of the M. pneumoniae targets. The ePlex Respiratory Pathogen Panel (RPP) assay detected 10/14 (71.4 %) of the L. pneumophila targets, 8/12 (66.7 %) of the B. pertussis positive samples and 13/15 (86.7 %) of the M. pneumoniae targets. CONCLUSIONS: No false-positive results were reported for all three bacterial pathogens by both assays. The clinical performance of both assays depended highly on the bacterial load in the sample and the type of specimen under investigation.

Measurement uncertainty evaluation and risk of false conformity assessment for microbial enumeration tests.

Microbial enumeration tests are widely used to assess the microbiological quality of non-sterile pharmaceutical products. Despite of all efforts to guarantee the reliability of microbial enumeration tests, there will always be an uncertainty associated with the measured values, which can lead to false conformity/non-conformity decisions. In this work, we evaluated the measurement uncertainty using a bottom-up approach and estimate the consumer's or producer's risk due to the measurement uncertainty. Three main sources of uncertainty were identified and quantified: dilution factor, plated volume, and microbial plate counts. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products. The contribution of dilution factor and plated volume uncertainties increase with an increase of measured value, while the contribution of microbial plate count uncertainty decreases with an increase of measured value. The overall uncertainty values were expressed as uncertainty factors, which provide an asymmetric 95% level confidence level of microbial load in pharmaceutical products. In addition, the risk of false conformity/non-conformity decisions due to measurement uncertainty was assess using Monte Carlo method. When the measured value is close to the upper specification limit and/or the measurement uncertainty is large, the risk of false conformity/non-conformity decisions may be significantly high. Thus, we conclude that the use of uncertainty factor in the conformity/non-conformity assessment is important to guarantee the reliability of microbial enumeration test results and to support decision-making.

Evaluation of a Novel Culture System for Rapid Pathogen Identification and Detection of Cephalosporin Resistance in Neonatal Gram-negative Sepsis at a Tertiary Referral Unit in Harare, Zimbabwe.

BACKGROUND: Neonatal sepsis accounts for a large proportion of neonatal deaths in sub-Saharan Africa. The lack of access to diagnostic testing and excessively long turnaround times to result contributes to delays in sepsis identification and initiation of appropriate treatment. This study aims to evaluate the novel InTrays COLOREX Screen and extended-spectrum beta-lactamase for rapid identification of bacterial pathogens causing sepsis and detection of resistance. METHODS: Neonates with suspected sepsis admitted to the Harare Central Hospital were prospectively enrolled. One blood culture was collected and incubated using the BacT/ALERT automated system. Positive blood cultures with potential pathogens identified by Gram stain were inoculated on the InTray COLOREX Screen and extended-spectrum beta-lactamase culture plates. RESULTS: A total of 216 neonates with suspected sepsis were recruited. Pathogens were isolated from blood cultures in 56 (25.9%) neonates of which 54 were Klebsiella pneumoniae. All K. pneumoniae were resistant to ceftriaxone and 53 (98%) were resistant to gentamicin. Sensitivity and specificity for ceftriaxone-resistant K. pneumoniae detection using InTrays were 100%. InTrays results were interpretable as early as 5-10 hours (median 7 hours, interquartile range 7-7) post blood culture positivity enabling rapid identification and notification of result and leading to a 60% reduction in time to result from blood culture collection. CONCLUSIONS: This study shows that the implementation of a novel culture method was feasible and reduced turnaround times for results by 60% compared with standard microbiologic techniques. An impact on patient outcomes and cost-effectiveness of this method needs to be demonstrated.

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA).

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers' safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log10eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

Establishing microbial composition measurement standards with reference frames.

Differential abundance analysis is controversial throughout microbiome research. Gold standard approaches require laborious measurements of total microbial load, or absolute number of microorganisms, to accurately determine taxonomic shifts. Therefore, most studies rely on relative abundance data. Here, we demonstrate common pitfalls in comparing relative abundance across samples and identify two solutions that reveal microbial changes without the need to estimate total microbial load. We define the notion of "reference frames", which provide deep intuition about the compositional nature of microbiome data. In an oral time series experiment, reference frames alleviate false positives and produce consistent results on both raw and cell-count normalized data. Furthermore, reference frames identify consistent, differentially abundant microbes previously undetected in two independent published datasets from subjects with atopic dermatitis. These methods allow reassessment of published relative abundance data to reveal reproducible microbial changes from standard sequencing output without the need for new assays.

Standardisation is necessary in urogenital and extragenital Chlamydia trachomatis bacterial load determination by quantitative PCR: a review of literature and retrospective study.

OBJECTIVES: Pathogen load has been linked to disease severity in patients infected with HIV, resulting in international standards to adequately and reproducibly quantify load. Chlamydia trachomatis (CT) load has been inconsistently linked to disease severity since extensive differences exist in quantification methods (14 methods in 28 articles). Differences include normalisation for human cell load due to CT's intracellular nature, despite the inability to distinguish inflammatory cells from epithelial cells with molecular techniques. We compared the human cell load in CT-positive men and women at the genital and anal site to a CT-negative control group to estimate the impact of inflammatory cells in these samples. METHODS: 188 women (tested at genital and anal site) and 519 men (207 tested at the anal site and 312 tested at the urogenital site) were included from our STI-clinic in the Netherlands. Specimens were self-collected vaginal swabs, anal swabs and urine samples. Quantitative-PCR targeting the HLA-gene quantified human cell load. Mann-Whitney-U-test was used for statistical analyses. RESULTS: The genital cell load had a similar range and median (6.5 log10) between CT-negative and CT-positive women . The urogenital cell load was significantly higher than the anal cell load (median 3.6 log10). The anal cell load was significantly higher in men with- than without anal CT infection (median 4.5 versus 3.9 respectively). The anal cell load is significantly higher in CT-positive men than in women. Both Neisseria gonorrhoeae-co-infections and reported anal intercourse significantly increased the human cell load in anal samples. CONCLUSION: Standardisation in CT load studies is necessary as current studies show 14 different quantification methods in 28 studies . In this study we demonstrate the inappropriateness of normalising the CT load for the human cell load using molecular techniques, as the presence of inflammatory cells cannot be excluded.

Concurrent Detection of Bacterial Pathogens in Hospital Roommates.

BACKGROUND: Some nurse-driven interventions have successfully reduced rates of healthcare-associated infections, though incidence remains unacceptably high. Bacterial contamination in patient rooms may be a source of exposure for patients and thus a target for future interventions; however, few studies have investigated the role of the patient room on organism acquisition. OBJECTIVES: The purpose of this study was to determine the incidence of concurrent detection of bacterial pathogens among patients sharing a hospital room. METHODS: We performed a retrospective network analysis using electronic administrative and clinical data collected from all patients admitted in 2006 through 2012 to four New York City hospitals, totaling 2,065 beds within 183 inpatient units. A computerized algorithm identified concurrent organism detection among roommates, defined as two patients who shared a room on at least 1 day and had a first positive culture for the same organism within 3 days following cohabitation. RESULTS: In total, 741,271 patient admissions were included. The algorithm identified 373 concurrent detection events: 158 (42%) in which the patients' first positive cultures were drawn after they were no longer sharing a room but within 3 days of cohabitation, 144 (39%) in which the patients' first positive cultures were drawn while they were still sharing a room but on different days, and 71 (19%) in which the patients' first positive cultures were drawn while they were sharing a room on the same day. DISCUSSION: Methods to improve environmental decontamination should be included as part of a comprehensive approach to infection prevention in hospitals. Nurses have an important role to play in the planning and implementation of interventions to reduce bioburden in the patient environment.

MC-Media Pad CC for Enumeration of Total Coliforms in a Variety of Foods.

BACKGROUND: Standard coliform count methods require the preparation of agar, the use of the pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad CC for the enumeration of coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. OBJECTIVE: Using a paired study design, the MC-Media Pad CC was compared to standard method ISO 4832:2006 for 10 matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice. METHODS: Each matrix was tested at three levels of coliform contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad CC and ISO 4832:2006 methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: The candidate and reference methods demonstrated SDs ranging from 0.027 to 0.264 and 0.025 to 0.157, respectively. The difference of means ranged from -0.015 to 0.381, showing no practical difference between the methods. The MC-Media Pad CC detected 58/62 inclusivity strains and correctly excluded 26/31 exclusivity organisms, similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. CONCLUSIONS: The results support the conclusions that the MC-Media Pad CC is a suitable alternative to the ISO 4832:2006 reference method for the matrixes examined and the data support AOAC Performance Tested MethodSM certification.

MC-Media Pad EC for Enumeration of Escherichia coli and Coliforms in a Variety of Foods.

BACKGROUND: Standard coliform count methods require preparation of agar, the use of pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad EC for enumeration of Escherichia coli and coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. OBJECTIVE: Using a paired study design, the MC-Media Pad EC was compared with standard method ISO 4832:2006. Ten matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice were evaluated in the study. METHODS: Each matrix was tested at three levels of contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad EC, ISO 4832:2006, and ISO 16649-2:2001 (Part 2) methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: The candidate and reference methods demonstrated standard deviations ranging from 0.034 to 0.188 and 0.028 to 0.181, respectively, for E. coli counts and 0.047-0.188 and 0.025-0.157, respectively, for total coliforms. The difference of means ranged from -0.025 to 0.331 for E. coli and from -0.037 to 0.372 for total coliforms, showing no practical difference between the methods. The MC-Media Pad EC detected 49/50 E. coli and 60/63 coliform inclusivity strains and correctly excluded 30/32 exclusivity organisms for E. coli and 24/31 exclusivity organisms for total coliforms, which was similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. CONCLUSIONS: The results support the conclusions that the MC-Media Pad EC is a suitable alternative to the ISO 4832:2006 and ISO 16649-2:2001 reference methods for the matrixes examined and the data support AOAC Performance Tested MethodSM certification. HIGHLIGHTS: The MC-Media Pad EC was approved for Performance Tested Method certification No. 011901.

Effectiveness of a neutral electrolysed oxidising water (NEOW) device in reducing Legionella pneumophila in a water distribution system: A comparison between culture, qPCR and PMA-qPCR detection methods.

Disinfection of hot water systems is critical for reducing Legionnaires' disease in high-risk buildings. The use of neutral electrolysed oxidising water (NEOW) is a promising method for the control of microorganisms in hot water systems. However, full-scale evaluations of the efficacy of NEOW devices to control Legionella pneumophila are currently lacking. The aim of this study was to assess the effectiveness of a NEOW device in reducing L. pneumophila in a hotel water network. Water samples (n = 67) were collected from different sites of a hotel distribution system before and after the installation of the NEOW device at the 1st, 4th, 8th and 12th week. Detection of L. pneumophila was performed comparing culture, qPCR and PMA-qPCR methods. Total bacterial counts (22 °C and 37 °C), Pseudomonas spp. and physico-chemical parameters were also monitored. The NEOW treatment resulted in a reduction of the amount of L. pneumophila positive samples (-32%) and of the number of heavily contaminated points (>104 CFU/L and >103 CFU/L) (-100% and -96%, respectively). Treatment maintained L. pneumophila at low levels (<102 CFU/L), which do not require specific intervention measures. The effectiveness of the disinfection system was also confirmed by PMA-qPCR (p < 0.001). The use of PMA resulted in a signal decrease in almost all samples upon the disinfection treatment. The NEOW disinfection device appears to be a promising approach to reduce the colonisation of hot water systems by L. pneumophila; however, further investigations are needed to ascertain its efficiency over longer time periods.

Effect of acids produced from carbohydrate metabolism in cryoprotectants on the viability of freeze-dried Lactobacillus and prediction of optimal initial cell concentration.

For the industrial production of probiotics powder, various sugars have been used as cryoprotectants to preserve probiotics during freeze-drying. Some of these sugars can be metabolized by Lactobacillus with the production of acids during the mix. In this study, we investigated the effect of acids on ATPase, ß-galactosidase, lactate dehydrogenase (LDH), integrity and fluidity of cell membrane and the survival rate of Lactobacillus during freeze-drying. In the presence of Lactobacillus, acids were produced from cryoprotectants containing fermentable sugars before freezing, resulting in a decrease in the pH of the bacterial suspension to below 5.0. During freeze-drying, the acids caused a loss of viability of Lactobacillus due to aggravated damage to ATPase, ß-galactosidase and cell membrane fluidity, but not LDH and cell membrane integrity. This finding implied that cryoprotectants that do not lead to the production of acids are effective in improving the survival rate of freeze-dried Lactobacillus. Here, a new formula was proposed for a protectant containing whey protein isolate (WPI) and rhamnose, which were not metabolized. In addition, linear-regression analyses were performed on the proportion of cryoprotectants (M) against cell paste (m), total cell count (N), total surface area (St) and total volume (Vt) of bacteria for 100% survival rate. The total surface areas of bacteria were found to be highly correlated with the amount of proposed cryoprotectant. The following prediction equation was established for the optimal initial cell concentration for a 100% survival rate of freeze-dried Lactobacillus: N (4πr2+2πl)=(0.66±0.03)M.

Mycobacterial Load Assay.

Tuberculosis is a difficult disease to treat, a process made more harder as tools to monitor treatment response only provide a result long after the patient has provided a sample. The mycobacterial load assay (MBLA) provides a simple molecular test to quantify and determine the viability of M. tuberculosis in human or other samples.

Optimization of cord blood unit sterility testing: impact of dilution, analysis delay, and inhibitory substances.

BACKGROUND: Different methods are used by cord blood banks to prepare samples for sterility testing. Suboptimal methods can result in the release of contaminated products. In our organization, samples are prepared by diluting the final product in RPMI-1640 medium. In this work, we have compared our method with different approaches to verify whether optimization should be sought. STUDY DESIGN AND METHODS: Cord blood units (n = 6 units per bacterial strain) characterized to contain inhibitory substances or not were inoculated (10 colony-forming units/mL) with Streptococcus agalactiae, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, or Bacteroides fragilis. After plasma and red blood cell removal, stem cell concentrates were diluted in RPMI-1640, thioglycollate, or the unit's plasma. These products, as well as final product, plasma, and red blood cell fractions, were held from 0 to 72 hours at 20 to 24°C before inoculation in culture bottles and detection using the BacT/ALERT 3D system. RESULTS: Dilution of cell concentrates in RPMI-1640 allowed bacterial detection in 93.3% of noninhibitory cord blood samples after a 24-hour storage period. Thioglycollate medium better promoted bacterial growth in inhibitory cord blood samples that were held for 72 hours before testing (66.7%) compared with RPMI-1640 (45.0%). Less than 33% of all spiked plasma samples were detected by the BacT/ALERT 3D system. CONCLUSION: Diluting cord blood samples in culture medium containing bacterial growth promoting substances is a suitable option for sterility testing, whereas the use of plasma should be proscribed, because it might lead to false-negative results. Because inhibitory substances affect bacterial growth, inoculation of culture bottles should be done rapidly after sample preparation.

Impact of enumeration method on diversity of Escherichia coli genotypes isolated from surface water.

There are numerous regulatory-approved Escherichia coli enumeration methods, but it is not known whether differences in media composition and incubation conditions impact the diversity of E. coli populations detected by these methods. A study was conducted to determine if three standard water quality assessments, Colilert® , USEPA Method 1603, (modified mTEC) and USEPA Method 1604 (MI), detect different populations of E. coli. Samples were collected from six watersheds and analysed using the three enumeration approaches followed by E. coli isolation and genotyping. Results indicated that the three methods generally produced similar enumeration data across the sites, although there were some differences on a site-by-site basis. The Colilert® method consistently generated the least diverse collection of E. coli genotypes as compared to modified mTEC and MI, with those two methods being roughly equal to each other. Although the three media assessed in this study were designed to enumerate E. coli, the differences in the media composition, incubation temperature, and growth platform appear to have a strong selective influence on the populations of E. coli isolated. This study suggests that standardized methods of enumeration and isolation may be warranted if researchers intend to obtain individual E. coli isolates for further characterization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterized the impact of three USEPA-approved Escherichia coli enumeration methods on observed E. coli population diversity in surface water samples. Results indicated that these methods produced similar E. coli enumeration data but were more variable in the diversity of E. coli genotypes observed. Although the three methods enumerate the same species, differences in media composition, growth platform, and incubation temperature likely contribute to the selection of different cultivable populations of E. coli, and thus caution should be used when implementing these methods interchangeably for downstream applications which require cultivated isolates.

Quantification of effective exoelectrogens by most probable number (MPN) in a microbial fuel cell.

The objective of this work was to quantify the number of exoelectrogens in wastewater capable of producing current in a microbial fuel cell by adapting the classical most probable number (MPN) methodology using current production as end point. Inoculating a series of microbial fuel cells with various dilutions of domestic wastewater and with acetate as test substrate yielded an apparent number of exoelectrogens of 17perml. Using current as a proxy for activity the apparent exoelectrogen growth rate was 0.03h(-1). With starch or wastewater as more complex test substrates similar apparent growth rates were obtained, but the apparent MPN based numbers of exoelectrogens in wastewater were significantly lower, probably because in contrast to acetate, complex substrates require complex food chains to deliver the electrons to the electrodes. Consequently, the apparent MPN is a function of the combined probabilities of members of the food chain being present.

A second look at the final number of cells in a fluctuation experiment.

In a fluctuation experiment, the number of cells existing in a culture immediately before plating (commonly known as Nt) varies across the parallel cultures. However, most existing mathematical models for fluctuation assay data do not recognize the variation in Nt. Despite repeated attempts in the past to integrate this source of variability in the estimation of microbial mutation rates, several questions of practical importance remain unanswered. The present investigation finds that the variation needs accounting for only when the coefficient of variation for Nt is large, and experimental data suggest that the coefficient of variation is often moderate or small. Moreover, an increase in the inoculum size can reduce the coefficient of variation. Through extensive simulation, several existing methods that accommodate the variation in Nt are compared. It was found that a newly devised likelihood method based on the existing gamma mixture model outperforms other existing methods. The investigation focuses on the estimation of mutation rates using the Lea-Coulson model, under which mutation is selectively neutral; however, the paper also explores the major findings' implications for the comparison of mutation rates using the likelihood ratio test, and for the estimation of mutation rates using the Mandelbrot-Koch model that allows for non-neutral mutations.

Comparative Evaluation of Inoculation of Urine Samples with the Copan WASP and BD Kiestra InoqulA Instruments.

This study evaluated the quantitative results from and quality of the inoculation patterns of urine specimens produced by two automated instruments, the Copan WASP and the BD InoqulA. Five hundred twenty-six urine samples submitted in 10-ml canisters containing boric acid were processed within 30 min on an InoqulA instrument plating 10 µl of specimen, and on two WASP instruments, one plating 1 µl of specimen (WASP-1), and the second plating 10 µl of WASP (WASP-10). All samples were incubated, analyzed, and digitally imaged using the BD Kiestra total lab automation system. The results were evaluated using a quantitative protocol and assessed for the presence or absence of ≥5 distinct colonies. Separate studies were conducted using quality control (QC) organisms to determine the relative accuracy of WASP-1, WASP-10, and InoqulA instruments compared to the results obtained with a calibrated pipette. The results with QC organisms were calculated as the ratios of the counts of the automated instruments divided by the counts for the calibrated pipette (the gold standard method). The ratios for the InoqulA instrument were closest to 1.0, with the smallest standard deviations indicating that compared to a calibrated pipette, the InoqulA results were more accurate than those with the WASP instrument. For clinical samples, the WASP instruments produced higher colony counts and more commensals than the InoqulA instrument, with differences most notable for WASP-1. The InoqulA instrument was significantly better at dispersing organisms with counts of ≥10(5) bacteria/ml of urine than were the WASP-1 and WASP-10 instruments (P < 0.05). Our results suggest that the InoqulA quantitative results are more accurate than the WASP results, and, moreover, the number of isolated colonies produced by the InoqulA instrument was significantly greater than that produced by the WASP instrument.