Comparative detection and recovery of Pseudomonas aeruginosa by membrane filtration and a Most Probable Number technique.

Two reference membrane filtration methods (Standard Methods: SM 9213E and U.S.EPA), the Most Probable Number method (Pseudalert®) and a membrane filtration method currently used by the Ontario Ministry of the Environment and Climate Change (MOECC) Laboratory Services Branch Etobicoke, Canada (Method E3371) were compared for the detection and recovery of Pseudomonas aeruginosa (P. aeruginosa) in sterile phosphate buffered dilution water (PBDW), un-spiked and spiked environmental water samples. The confirmation of presumptive-positive P. aeruginosa colonies was performed by API®20NE and on Milk Agar. Detection and recoveries were compared by determining the sensitivity and specificity of the methods and the performance of the methods was evaluated for statistical differences using ANOVA. The recovery of P. aeruginosa from PBDW spiked at a level of 100CFU/100mL was significantly higher (p<0.05) with Pseudalert® compared to the other methods. However, there were no significant differences (p>0.5) between all four methods when spiked at 5 and 40CFU/100mL. In the analysis of 61 environmental surface water samples, the MPN method (Pseudalert®) demonstrated the highest sensitivity (100%) while the Standard Method-SM9213E had the lowest sensitivity (3%). The U.S.EPA method and SM9213E demonstrated the highest specificity (100%) while Pseudalert® had the lowest (71.0%). Pseudalert® was able to detect P. aeruginosa in samples with higher amount of suspended solids as compared to other MF methods. Analysis of 24 environmental surface water samples (previously found to be negative for P. aeruginosa), which were spiked with P. aeruginosa at 5CFU/100mL, showed different sensitivities in recovery including Pseudalert® (100%), MOECC E3371 (92%), U.S.EPA (33%) and SM 9213E (33%). The highest mean recovery was observed with Pseudalert® (5.3 MPN/100mL) and the least with the U.S.EPA method (0.4CFU/100mL). Pseudalert® demonstrated improved performance in the detection and recovery of P. aeruginosa over SM9213E, U.S.EPA and MOECC E3371 in terms of sensitivity. However, Pseudalert® reported the highest amount of false positive results compared to the other three MF methods. The addition of a confirmation step with Milk Agar eliminated the false positive results. Therefore, this confirmatory step is recommended in order to increase the specificity of the Pseudalert® method.

A skin lesion that catches the eye.

Primary cutaneous gamma-delta T-cell lymphoma (PCGD-TCL) is rare and only represents 1% of all cutaneous T-cell lymphomas. To our knowledge, only 40 cases have been described. It often presents with generalised skin lesions, preferentially affecting the extremities. There is a well-documented association with haemophagocytic syndrome. Treatment is difficult since PCGD-TCL is often resistant to chemotherapy and radiotherapy. Most case reports describe an aggressive clinical course with an estimated mean survival of 15 months. We present a 72-year-old female patient with stage IV primary cutaneous gamma-delta T-cell lymphoma. Our patient presented with fever, night sweats and multiple skin lesions (figure 1). Computed axial tomography of chest and abdomen revealed multiple solid nodular lesions in both kidneys. During admission a subconjunctival lesion appeared and progressed rapidly (figure 2). Histopathological examination of skin biopsy revealed infiltration of atypical lymphocytes with hyperchromatic irregular nuclei. Immunophenotyping pattern of skin biopsy was compatible with PCGD-TLC. Clonal gamma-delta T-cells were also detected by immunohistochemical analysis of peripheral blood and bone marrow. Polymerase chain reaction amplification revealed clonal rearrangement of the T-cell receptor gamma chain gene. These findings together were consistent with stage IV primary cutaneous gamma-delta T-cell lymphoma. The rapid progression of the subconjunctival extra-nodal manifestation is characteristic for the aggressive course of this lymphoma. Our patient was treated with two cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone). However, her clinical condition deteriorated rapidly. She declined further therapy and died within three months of initial presentation.

Detection and enumeration of E. coli O157:H7 in water samples by culture and molecular methods.

The performances of three chromogenic agars were evaluated for the recovery of Escherichia coli O157:H7 from spiked dechlorinated tap, ground and surface water, and treated drinking water samples. The chromogenic agars: ChromAgar O157 (CHROM), Rainbow Agar O157 (RB) and HiCrome EC O157 (HC) were compared to cefixime-tellurite Sorbitol MacConkey (CT-SMAC), commonly used for the isolation of E. coli O157:H7. Confirmation of suspect E. coli O157:H7 colonies were performed by colony real-time PCR (C-RTi-PCR) based on the presence of Shiga-toxin genes (stx1 and stx2). Recovery of inoculated E. coli O157:H7 from dechlorinated tap water indicated that RB and CHROM agars demonstrated improved recovery when compared to HC or CT-SMAC. There was a significant drop in recovery on all agars tested after 120h (day 5). Twenty dechlorinated tap and/or treated drinking water samples were inoculated with a pure culture of E. coli O157:H7 (ATCC 43894), and a mixed culture of E. coli O157:H7 (ATCC 43894), E. coli strain K-12, and Enterococcus faecalis (ATCC 063589). After a 48-hour holding time, the recovery using CHROM (99%) and HC (12%) from samples contaminated with the pure culture were found to be significantly different (p<0.05). Recovery results using CHROM (39%) and CT-SMAC (32%) from samples contaminated with the mixed culture after a 48-hour holding time were not significantly different (p>0.05). Analysis by C-RTi-PCR of forty five environmental water samples (surface, sewage, and final effluents) which were negative for E. coli O157:H7 showed an incidence of false suspect positive colonies of 38% (CHROM), 53% (RB), 58% (HC), and 91% (CT-SMAC). Further analysis of eight of the environmental samples inoculated with E. coli (ATCC 43894) showed 100% recovery when utilizing CHROM, 50% when using RB and 40% when using HC. In addition, the C-RTi-PCR positive confirmation rate was 100% for CHROM and HC and 65% for RB. CHROM demonstrated improved recovery of E. coli O157:H7 over RB, HC, and CT-SMAC in terms of sensitivity and specificity.

6q deletion in Waldenström macroglobulinemia is associated with features of adverse prognosis.

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenström macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of beta2-microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q- among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series.

Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites.

In B-cell lymphomas, loss of human leukocyte antigen (HLA) class I and II molecules might contribute to immune escape from CD8(+) and CD4(+) cytotoxic T cells, especially because B cells can present their own idiotype. Loss of HLA expression and the possible underlying genomic alterations were studied in 28 testicular, 11 central nervous system, and 21 nodal diffuse large B-cell lymphomas (DLCLs), the first two sites are considered as immune-privileged sites. The analysis included immunohistochemistry, loss of heterozygosity analysis, and fluorescent in situ hybridization (FISH) on interphase cells and isolated DNA fibers. Total loss of HLA-A expression was found in 60% of the extranodal cases and in 10% of the nodal cases (P <.01), whereas loss of HLA-DR expression was found in 56% and 5%, respectively (P <.01). This was accompanied by extensive loss of heterozygosity within the HLA region in the extranodal DLCLs. In 3 cases, retention of heterozygosity for D6S1666 in the class II region suggested a homozygous deletion. This finding was confirmed by interphase FISH that showed homozygous deletions in the class II genes in 11 of the 18 extranodal lymphomas but in none of the 7 nodal DLCLs (P <.001). Mapping by fiber FISH showed variable deletions that always included HLA-DQ and HLA-DR genes. Hemizygous deletions and mitotic recombinations often involving all HLA genes were found in 13 of 18 extranodal and 2 of 7 nodal lymphomas. In conclusion, a structural loss of HLA class I and II expression might help the B-cell lymphoma cells to escape from immune attack.

Assessment of 1,2-dimethylhydrazine in bone marrow micronucleus assay: variations in protocol and response.

The effect of variations in experimental protocol on the assessment of the genotoxicity of 1,2-dimethylhydrazine (DMH) in the bone marrow micronucleus assay was determined. The incidence of micronuclei (MN) in the bone marrow of CBA mice treated with DMH (either intraperitoneally (i.p.) or orally) was found to be significantly greater than that observed in C57B1/6J mice using the same dose and dosing regimen. With i.p. injection, DMH, at doses of 20 and 50 mg/kg, was found to be positive in the bone marrow MN test in CBA mice only. In C57B1/6J mice, DMH (i.p.) was found to be positive at only the 50 mg/kg dose. With oral administration, DMH was positive in the MN test only at the 50 mg/kg dose and only in CBA mice. No significant difference in the percentage of MN was observed when 300, 500, or 1,000 polychromatic erythrocytes (PCEs) were scored following a single treatment of DMH. Cyclophosphamide (CY) was found to induce a dose-dependent increase in the percentage of MN observed in the bone marrow of C57B1/6J mice. DMH tested positive in the colon nuclear aberration (NA) assay in both strains of mice using both i.p. and oral routes of administration, although C57B1/6J mice were found to be more sensitive than CBA mice. No significant difference was observed regarding the percentage of NAs observed in the colon between mice injected i.p. or orally gavaged.

Comparison of the activity of topically applied pesticides and the herbicide 2,4-D in two short-term in vivo assays of genotoxicity in the mouse.

Genotoxicity of eight topically applied compounds was determined using the bone marrow micronucleus (MN) test and hair follicle nuclear aberration (NA) assay in CD1 mice. Twenty-four hours after a single treatment, cyclophosphamide (CY), applied at doses corresponding to 1/4, 1/8, 1/16, and 1/32 of the published dermal LD50, and N-methyl-N-nitrosourea (MNU), applied at 1/4, 1/8, and 1/16 of the published dermal LD50, were found to increase the incidence of NA in a dose-dependent manner. The frequency of MN was significantly increased only at the highest dose of CY. Using the same protocol, six pesticides applied in dimethyl sulfoxide (DMSO) at doses of 1/8, 1/16, and 1/32 of the dermal LD50 were investigated. Aminocarb and chlordane induced a dose-dependent increase in the frequency of NA, while there was an observed increase in NA incidence at only the highest doses of dichlorvos (DDVP), 4,4'-DDT (DDT), and 2,4-dichlorophenoxyacetic acid (2,4-D). No effect was observed with fenitrothion on nuclear aberrations in hair follicles. Except for the highest dose of chlordane, none of the pesticides tested positive in the bone marrow micronucleus test. Serum cholinesterase levels were reduced to 70 +/- 4.7% of the DMSO control level with DDVP, 57 +/- 8.2% with aminocarb, and 60.3 +/- 4.8% with fenitrothion, indicating some systemic activity with these topically applied agents. The data suggest that aminocarb, chlordane, DDVP, DDT, and 2,4-D are genotoxic as determined by the NA assay and that this assay may be more useful in detecting topically applied genotoxic agents than the more often used bone marrow micronucleus test.

Nongenotoxicity of minoxidil in murine hair follicles as determined by the nuclear aberration assay.

A 1-cm2 area on the back of CD1 mice was prepared for topical application of minoxidil, N-methyl-N-nitrosourea (MNU), or cyclophosphamide (CY) by clipping or plucking hair from a patch of skin. Plucking stimulates hair follicle cell division while clipping does not. Minoxidil was topically administered for 8 consecutive days. CY or MNU was administered topically once on the eighth day postplucking. The incidence of nuclear aberrations and mitotic figures were measured in hair follicles while frequency of micronuclei and the ratio of RBC/PCE were measured in the bone marrow. Results with minoxidil showed no increase in either nuclear aberrations in the hair follicle or micronuclei in the bone marrow. These results suggest that topically applied minoxidil is not genotoxic. In contrast, a dose-dependent effect of MNU on the incidence of nuclear aberrations in the hair follicle was seen. CY induced a dose-dependent increase in the incidence of micronuclei in the bone marrow and in nuclear aberrations in the hair follicle after topical application. Minoxidil applied to clipped mice significantly increased the incidence of mitotic figures above that seen in both the clipped and plucked controls. This suggests that minoxidil is a mitogenic agent in the hair follicle. These findings are consistent with the success of topically applied minoxidil in the treatment of alopecia areata.