A review of the factors affecting sunlight inactivation of micro-organisms in waste stabilisation ponds: preliminary results for enterococci.

Waste stabilisation ponds (WSP) are efficient, cost-effective methods of treating wastewater in rural and remote communities in Australia. It is recognised that sunlight plays a significant role in their disinfection, however, due to the poor penetration of light in turbid waters it has been hypothesised that other mechanisms may also contribute to disinfection in WSPs. To date, studies have reported various and conflicting results with regards to the relative contributions of UVA, UVB, PAR and environmental factors including pH, DO and photo-sensitisers on micro-organism disinfection. Initially we investigated the role of these environmental factors on the solar disinfection of enterococci in buffered distilled water to control for potential confounding factors within the wastewater. Die-off rate constants were measured, in sterile buffered distilled water at varying pH and dissolved oxygen concentrations, for enterococci irradiated with UVA and UVB. Enterococci were found to be predominantly inactivated by UVB (p<0.001), however, UVA was also observed to increase inactivation rates relative to the dark control (p<0.001). DO and pH were found to have no effect on inactivation rate when enterococci were irradiated with UVB (p>0.05), however, when irradiated with UVA, both DO and pH were observed to further increase inactivation rates (p<0.01).

Detection of Chlamydia trachomatis with fluorescent monoclonal antibody.

Commercial kits of fluorescein-labelled monoclonal antibody against Chlamydia trachomatis have been evaluated (1) as an alternative to Giemsa staining for detection of chlamydia inclusions in cell culture and (2) for direct detection of chlamydia in conjunctival, urethral and cervical smears. The inclusion-detection kit (Micro Trak Culture Confirmation Test) was tested on 270 cultures and was found to be highly sensitive, detecting all 16 Giemsa-positive specimens plus an additional 3 that were negative by Giemsa. It was also superior to Giemsa-staining in terms of simplicity of use, ease of detection, and readability with toxic specimens. The direct detection kit (Micro Trak Direct Specimen Test) gave results which agreed with the culture result for all 33 genital tract specimens and for 32 of 34 conjunctival specimens tested in parallel. The kit is considered to be a valuable test for diagnosis of chlamydial infection by laboratories lacking adequate tissue-culture facilities.

Rapid diagnosis of viral infections with fluorescent antisera.

Viral immunofluorescence tests were performed with commercial antisera on 1046 specimens of respiratory, conjunctival corneal and dermal origin to compare the diagnostic effectiveness of direct examination by immunofluorescence with culture. The fluorescence assays were found to be highly sensitive for respiratory syncytial and measles virus in nasopharyngeal aspirates from children, but less satisfactory for other respiratory viruses. The test detected 38% of the adenovirus positives from conjunctival specimens and 67% of the combined herpes simplex virus positives from the latter 3 groups. Despite the reduced sensitivity of fluorescence for some viruses, a case is presented for more widespread use of fluorescence in routine microbiology laboratories because of its simplicity, speed and cost-effectiveness compared with culture which is labour-intensive and time consuming.

Development of an enzyme immunoassay to detect antibody to Chlamydia pneumoniae strain TWAR and its application in a limited seroepidemiological survey.

A non-competitive enzyme immunoassay (EIA) was developed to detect IgG antibody to the recently-described species Chlamydia pneumoniae strain TWAR. Purified elementary bodies of the organism were used as capture antigen. Cross-reacting antibody to Chlamydia trachomatis was detected in the assay by running a parallel EIA for IgG antibody to C. trachomatis. The C. pneumoniae assay was validated by comparing the results on 60 selected sera with those obtained with the microimmunofluorescence test. The comparison indicated that the EIA had a sensitivity of 88%, specificity of 100% and overall correlation with micro-immunofluorescence of 93%. The assay was applied to 352 sera from 3 populations within Australasia in a limited survey to determine the extent of exposure to this organism. Prevalence rates of up to 55% were found, suggesting that a significant amount of respiratory disease in the region may be due to C. pneumoniae strain TWAR.

Comparison of antigen detection, polymerase chain reaction and culture for detection of Chlamydia trachomatis in genital infection.

An in-house polymerase chain reaction (PCR) test using ribosomal RNA gene primers was compared with chlamydia antigen detection (DIF) and culture for the detection of Chlamydia trachomatis. Five hundred and forty-eight fresh (unstored) genital swabs and 174 urines (collected at the same time) from patients attending a sexually-transmitted diseases clinic were examined. PCR, DIF and culture detected chlamydia in 43, 35 and 42 swabs respectively from the 43 resolved positive cases. The specificity on the resolved negative specimens was 100% for each of the tests. From the urines, PCR and DIF detected the organism in 16 and 15 cases respectively of the 23 resolved positive males tested but in only 2 and 3 cases respectively of the 9 resolved positive females tested. Specificities were 100% in all cases. Both of the non-culture tests manifested problems with urine due to inhibitory activity (in PCR test) or excessive debris (in DIF test) in about 5% of the specimens. Culture of the urines yielded sensitivities of 40% in the males and 22% in the females. Overall PCR was more sensitive than either culture or DIF on both urethral and cervical swabs and urines. The urines yielded less than three-quarters the number of positives that was obtained from the swabs and were considered to be an unsatisfactory specimen for chlamydial diagnosis. It is concluded that PCR is a satisfactory alternative to culture on genital swabs and may be preferable in situations where the viability of the organisms is in question. DIF remains useful because of its speed and simplicity but is insufficiently sensitive to be relied upon by itself.

Enzyme-linked immunosorbent assay for detection of Haemophilus influenzae type b antigen.

An enzyme-linked immunosorbent assay for the detection of Haemophilus influenzae type b antigen was developed. It was able to detect purified polyribose phosphate at concentrations of greater than or equal to 1 ng/ml in cerebrospinal fluid. This was 50 times more sensitive than counterimmunoelectrophoresis with the same antiserum. The sensitivity for polyribose phosphate in urine was similar, but that in serum was about 10 times less. Nonspecific reactions were observed with blood-stained cerebrospinal fluid and some sera. These were differentiated from true positive reactions by a blocking test with unconjugated immune serum. A wide range of organisms was tested for cross-reactivity in the assay. With the exception of a protein A-rich strain of Staphylococcus aureus, they gave absorbances of < 8% of that of the homologous system. In a series of five cases of proven H. influenzae type b meningitis, the sensitivity of the assay with cerebrospinal fluid was confirmed to be at least 2(5) times greater than that of counterimmunoelectrophoresis. The results indicate that the enzyme-linked immunosorbent assay is highly sensitive and specific in detecting H. influenzae type b antigen. The necessity to perform the blocking assay on all sera limits its usefulness for the examination of these specimens. However, it should prove valuable for the detection of the antigen in cerebrospinal fluid and urine.

Adhesion molecule expression on epithelial cells infected with respiratory syncytial virus.

Respiratory epithelium is both a target and an effector of airway inflammation. Adhesion molecules on epithelium play an important role in a variety of airway diseases. Respiratory syncytial virus (RSV) is the most important pathogen for airway diseases in infants. The expression of adhesion molecules on epithelium in RSV infection, however, is unclear. The expression of selected adhesion molecules and major histocompatibility complex (MHC) class I and II antigens on a human alveolar type II epithelial cell line (A549) infected with RSV was investigated by means of flow cytometry and immunocytochemistry. The results showed that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were expressed on A549 cells at a low level. E-cadherin and MHC class I antigen were constitutively expressed on the cells. RSV infection of A549 cells significantly upregulated the expression of ICAM-1, VCAM-1 and MHC class I and II antigens on these cells. RSV infection also altered the expression of E-cadherin on A549 cells. Immunostaining showed that E-cadherin was mainly upregulated around or in RSV-induced giant cells. These data suggest that respiratory syncytial virus infection of respiratory epithelial cells enhances the expression of adhesion molecules and major histocompatibility complex antigens. These changes may play an important role in the pathophysiology of respiratory syncytial virus disease.