CMOS based image cytometry for detection of phytoplankton in ballast water.

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

An image cytometer based on angular spatial frequency processing and its validation for rapid detection and quantification of waterborne microorganisms.

We introduce a new image cytometer design for the detection of very small particulates and demonstrate its capability in water analysis. The device is a compact microscope composed of off-the-shelf components, such as a light emitting diode (LED) source, a complementary metal-oxide-semiconductor (CMOS) image sensor, and a specific combination of optical lenses that allow, through an appropriate software, Fourier transform processing of the sample volume. Waterborne microorganisms, such as Escherichia coli (E. coli), Legionella pneumophila (L. pneumophila) and phytoplankton, are detected by interrogating the volume sample either in a fluorescent or label-free mode, i.e. with or without fluorescein isothiocyanate (FITC) molecules attached to the micro-organisms, respectively. We achieve a sensitivity of 50 cells per ml, which can be further increased to 0.2 cells per ml by pre-concentrating an initial sample volume of 500 ml with an ad hoc fluidic system. We also prove the capability of the proposed image cytometer of differentiating microbiological populations by size with a resolution of 3 µm and operating in real contaminated water.

Quantitative detection of Helicobacter pylori in water samples by real-time PCR amplification of the cag pathogenicity island gene, cagE.

AIMS: A new real-time PCR assay that simultaneously amplifies a 102-bp fragment of the cagE gene from Helicobacter pylori and a new internal positive control containing a specific sequence of the gyrB gene from Aeromonas hydrophila, was developed and validated for the detection of H. pylori in environmental samples. METHODS AND RESULTS: The specificity, limits of detection and quantification, repeatability, reproducibility, and accuracy of the method were calculated. The resulting values confirmed the applicability of the method for the quantitative detection of H. pylori. The feasibility of the method was also evaluated by testing 13 pyloric antrum-positive biopsies and 69 water samples, including potable (10), surface (19) and wastewater (40) matrices. The results showed that all the biopsies and 3 of the 40 wastewater samples analysed were positive. CONCLUSIONS: This real-time PCR method provides a sensitive, specific, and accurate method for the rapid quantification of H. pylori in environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR diagnostic system proposed in this work, provides a suitable tool for the quantitative detection of H. pylori in environmental samples and can be useful for verifying the role of water as a potential route of its transmission.

Three-dimensional facial morphology following surgical repair of unilateral cleft lip and palate in patients after nasoalveolar molding.

OBJECTIVE: To evaluate three-dimensional (3D) facial morphology in patients surgically corrected for unilateral cleft lip and palate (UCLP) following pre-surgical nasoalveolar molding (NAM). DESIGN: Prospective, longitudinal study. Digital stereophotogrammetry was used to capture 3D facial images, and x, y, and z coordinates of five landmarks were digitized to compute mean morphologies. The sample comprised 15 patients with left UCLP and 10 matched control subjects. Facial form differences at age 37 weeks, using principal components analysis and finite-element scaling analysis (FESA) were assessed. RESULTS: Using the first two principal components, which accounted for 63% of the total shape-change, UCLP and control groups showed similar distributions in the modal space (p > 0.05). For the UCLP group, the mean 3D facial form was smaller and less protrusive when superimposed on the non-cleft mean. Using FESA, reductions in facial volume were found in the UCLP group, involving the columella (29%), labial tubercle (51%), lower lip (29%) and lateral aspects of the face (19%). The UCLP group also showed increases in size above the tip of the nose (25%) and laterally to the columella directly below the nares (29%). CONCLUSIONS: Following surgical repair of UCLP in patients previously treated with NAM, 3D facial morphology was virtually indistinguishable from the non-cleft mean. Clinically, the apparent improvement in the facial soft tissues may mask dysmorphic skeletal growth, and further studies are required to characterize the underlying bony changes associated with the soft tissue changes reported here.

Validation of a new seminested PCR-based detection method for Legionella pneumophila.

We report a new seminested PCR method for detection of Legionella pneumophila based on the simultaneous amplification of a 387 bp fragment of the dotA gene and a 827 bp recombinant fragment that serves as an internal positive control. This new detection system was validated and its specificity and detection limit were determined. Parallel analysis of 90 environmental samples to compare this method, a real-time PCR method and the standard culture isolation method, demonstrated that this seminested method is useful for the study of L. pneumophila.

A simple and cost-effective method for the quantification of total coliforms and Escherichia coli in potable water.

In this study, a new simple and cost-effective method for the study of total coliforms and Escherichia coli in potable water, combining the use of lactose TTC agar and TBX agar, was developed and compared with methods using Chromocult agar and coli ID. The statistical analysis showed no significant difference and a good correlation (R(2)) between the three methods.

Evaluation of the MicroFoss system for the analysis of Escherichia coli in water.

In this study, the performance of the MicroFoss system (Foss, Spain) for the enumeration of Escherichia coli in water samples was evaluated. One hundred and eighty-five samples were analysed both by MicroFoss assay and culture isolation on Tryptone-Bile X-glucuronide agar (TBX), and the correlation coefficient obtained was 0.92. The analysis of 28 new samples using both methods showed a statistically significant relationship at the 99.5% confidence level between log colony forming units obtained by MicroFoss assay and those obtained using growth on TBX agar. Nevertheless, when the level of sample contamination was low, the variability was high. In conclusion, the MicroFoss system is a rapid and simple alternative method for the enumeration of E. coli in water although discordance between the results using these methods in samples with low counts could limit its use for the study of clean water such as potable water.

Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene.

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.

The phylogenetic relationships of all known species of the genus Aeromonas, and especially Aeromonas bestiarum and Aeromonas salmonicida, were investigated on 70 strains using the rpoD sequence, which encodes the sigma70 factor. This analysis was complemented with the sequence of gyrB, which has already proven useful for determining the phylogenetic relationships in the genus. Nucleotide sequences of rpoD and gyrB showed that both genes had similar substitution rates (< 2 %) and a similar number of variable positions (34 % for rpoD versus 32 % for gyrB). Strain groupings by analysis of rpoD, gyrB and a combination of both genes were consistent with the taxonomic organization of all Aeromonas species described to date. However, the simultaneous analysis of both clocks improved the reliability and the power to differentiate, in particular, closely related taxa. At the inter-species level, gyrB showed a better resolution for differentiating Aeromonas sp. HG11/Aeromonas encheleia and Aeromonas veronii/Aeromonas culicicola/Aeromonas allosaccharophila, while rpoD more clearly differentiated A. salmonicida from A. bestiarum. The analysis of rpoD provided initial evidence for clear phylogenetic divergence between the latter two species.

Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences.

The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase. Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions. The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria. This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date. In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species. The present analysis was useful for the elucidation of reported discrepancies between different DNA-DNA hybridization sets. Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains. In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas.

Prior genital tract infection with a murine or human biovar of Chlamydia trachomatis protects mice against heterotypic challenge infection.

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar of Chlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars of C. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murine C. trachomatis induces immunity against challenge with either of two human biovars.

Determination of mycobacterial antigens in sputum by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) was examined for its usefulness in detecting mycobacterial antigens in sputum. A double-antibody sandwich procedure was set up by using a commercially available hyperimmune serum directed against Mycobacterium bovis, BCG. The ELISA was able to detect 10 ng of protein per ml of BCG sonic extract. The system also clearly distinguished Mycobacterium tuberculosis organisms from Mycobacterium avium and Mycobacterium kansasii organisms. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smear-negative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. This approach offers promise as an aid in the presumptive differentiation of nontuberculous mycobacteria from the M. tuberculosis complex.