Comparing the Resistance, Resilience, and Stability of Replicate Moving Bed Biofilm and Suspended Growth Combined Nitritation-Anammox Reactors.

Combined partial nitritation-anammox (PN/A) systems are increasingly being employed for sustainable removal of nitrogen from wastewater, but process instabilities present ongoing challenges for practitioners. The goal of this study was to elucidate differences in process stability between PN/A process variations employing two distinct aggregate types: biofilm [in moving bed biofilm reactors (MBBRs)] and suspended growth biomass. Triplicate reactors for each process variation were studied under baseline conditions and in response to a series of transient perturbations. MBBRs displayed elevated NH4+ removal rates relative to those of suspended growth counterparts over six months of unperturbed baseline operation but also exhibited significantly greater variability in performance. Transient perturbations led to strikingly divergent yet reproducible behavior in biofilm versus suspended growth systems. A temperature perturbation prompted a sharp reduction in NH4+ removal rates with no accumulation of NO2- and rapid recovery in MBBRs, compared to a similar reduction in NH4+ removal rates but a high level of accumulation of NO2- in suspended growth reactors. Pulse additions of a nitrification inhibitor (allylthiourea) prompted only moderate declines in performance in suspended growth reactors compared to sharp decreases in NH4+ removal rates in MBBRs. Quantitative fluorescence in situ hybridization demonstrated a significant enrichment of anammox in MBBRs compared to suspended growth reactors, and conversely a proportionally higher AOB abundance in suspended growth reactors. Overall, MBBRs displayed significantly increased susceptibility to transient perturbations employed in this study compared to that of suspended growth counterparts (stability parameter), including significantly longer recovery times (resilience). No significant difference in the maximal impact of perturbations (resistance) was apparent. Taken together, our results suggest that aggregate architecture (biofilm vs suspended growth) in PN/A processes exerts an unexpectedly strong influence on process stability.

Review on the fate of organic micropollutants in wastewater treatment and water reuse with membranes.

A brief review of the fate of micropollutants in membrane-based wastewater treatment due to sorption, stripping, biological degradation/transformation and membrane separation is discussed, to give an overview of these technologies due to the growing importance for water reuse purposes. Compared with conventional activated sludge treatment (CAS) micropollutant removal in membrane bioreactor (MBR) is slightly improved due to complete suspended solids removal and increased sludge age. For discharge to sensitive receiving waters advanced treatment, such as post-ozonation or activated carbon adsorption, is recommended. In water reuse plants nanofiltration (NF) and reverse osmosis (RO) efficiently reject micropollutants due to size exclusions as well as electrostatic and hydrophobic effects reaching potable quality. To remove micropollutants fully, additionally post-ozone or the addition of powdered activated carbon (PAC) have to be applied, which in parallel also reduce NDMA precursors. The concentrate has to be treated if disposed to sensitive receiving waters due to its high micropollutant concentration and ecotoxicity potential. The present review summarizes principles and capabilities for the most important membrane-based applications for wastewater treatment, i.e. porous membranes in MBRs (micro- or ultrafiltration) and dense membrane applications (NF and RO) for water reuse.

Removal of micropollutants in municipal wastewater treatment plants by powder-activated carbon.

Micropollutants (MP) are only partly removed from municipal wastewater by nutrient removal plants and are seen increasingly as a threat to aquatic ecosystems and to the safety of drinking water resources. The addition of powder activated carbon (PAC) is a promising technology to complement municipal nutrient removal plants in order to achieve a significant reduction of MPs and ecotoxicity in receiving waters. This paper presents the salient outcomes of pilot- and full-scale applications of PAC addition in different flow schemes for micropollutant removal in municipal wastewater treatment plants (WWTPs). The sorption efficiency of PAC is reduced with increasing dissolved organic carbon (DOC). Adequate treatment of secondary effluent with 5-10 g DOC m(-3) requires 10-20 g PAC m(-3) of effluent. Counter-current use of PAC by recycling waste PAC from post-treatment in a contact tank with an additional clarifier to the biology tank improved the overall MP removal by 10 to 50% compared with effluent PAC application alone. A dosage of 15 g PAC m(-3) to a full-scale flocculation sand filtration system and recycling the backwash water to the biology tank showed similar MP elimination. Due to an adequate mixing regime and the addition of adapted flocculants, a good retention of the fine fraction of the PAC in the deep-bed filter were observed (1-3 g TSS m(-3); TSS: total suspended solids). With double use of PAC, only half of the PAC was required to reach MP removal efficiencies similar to the direct single dosage of PAC to the biology tank. Overall, the application of PAC in WWTPs seems to be an adequate and feasible technology for efficient MP elimination (>80%) from wastewater comparable with post ozonation.

Successful year-round mainstream partial nitritation anammox: Assessment of effluent quality, performance and N2O emissions.

For two decades now, partial nitritation anammox (PNA) systems were suggested to more efficiently remove nitrogen (N) from mainstream municipal wastewater. Yet to date, only a few pilot-scale systems and even fewer full-scale implementations of this technology have been described. Process instability continues to restrict the broad application of PNA. Especially problematic are insufficient anammox biomass retention, the growth of undesired aerobic nitrite-oxidizers, and nitrous oxide (N2O) emissions. In this study, a two-stage mainstream pilot-scale PNA system, consisting of three reactors (carbon pre-treatment, nitritation, anammox - 8 m3 each), was operated over a year, treating municipal wastewater. The aim was to test whether both, robust autotrophic N removal and high effluent quality, can be achieved throughout the year. A second aim was to better understand rate limiting processes, potentially affecting the overall performance of PNA systems. In this pilot study, excellent effluent quality, in terms of inorganic nitrogen, was accomplished (average effluent concentrations: 0.4 mgNH4-N/L, 0.1 mgNO2-N/L, 0.9 mgNO3-N/L) even at wastewater temperatures previously considered problematic (as low as 8 °C). N removal was limited by nitritation rates (84 ± 43 mgNH4-N/L/d), while surplus anammox activity was observed at all times (178 ± 43 mgN/L/d). Throughout the study, nitrite-oxidation was maintained at a low level (<2.5% of ammonium consumption rate). Unfortunately, high N2O emissions from the nitritation stage (1.2% of total nitrogen in the influent) were observed, and, based on natural isotope abundance measurements, could be attributed to heterotrophic denitrification. In situ batch experiments were conducted to identify the role of dissolved oxygen (DO) and organic substrate availability in N2O emission-mitigation. The addition of organic substrate, to promote complete denitrification, was not successful in decreasing N2O emission, but increasing the DO from 0.3 to 2.9 mgO2/L decreased N2O emissions by a factor of 3.4.

Successful mainstream nitritation through NOB inactivation.

The development of new wastewater treatment processes can assist in reducing the impact of wastewater treatment on the environment. The recently developed partial nitritation anammox (PNA) process, for example, consumes less energy for aeration and reduces nitrate in the effluent without requiring additional organic carbon. However, achieving stable nitritation (ammonium oxidation; NH4+ → NO2-) at mainstream conditions (T = 10-25 °C, C:N > 10, influent ammonium < 50 mgNH4-N/L and effluent < 1 mgNH4-N/L) remains challenging. This study explores the potential and mechanism of nitrite-oxidizing bacteria (NOB) suppression in a bottom-fed sequencing batch reactor (SBR). Two bench-scale (11 L) reactors and a pilot-scale reactor (8 m3) were operated for over a year and were fed with organic substrate depleted municipal wastewater. Initially, nitratation (nitrite oxidation; NO2- → NO3-) occurred occasionally until an anaerobic phase was integrated into the operating cycle. The introduction of the anaerobic phase effectively suppressed the regrowth of NOB while nitritation was stable over 300 days, down to 8 °C and at ammonium influent concentrations < 25 mgNH4-N/L. Batch experiments and process data revealed that parameters typically affecting NOB growth (e.g., dissolved oxygen, alkalinity, trace elements, lag-phase after anoxia, free nitrous acid (FNA), free ammonia (FA), pH, sulfide, or solids retention time (SRT)) could not fully explain the suppression of nitratation. Experiments in which fresh nitrifying microbial biomass was added to the nitritation system indicated that NOB inactivation explained NOB suppression better than NOB washout at high SRT. This study concludes that bottom-fed SBRs with anaerobic phases allow for stable nitritation over a broad range of operational parameters. Coupling this type of SBR to an anammox reactor can enable efficient mainstream anammox-based wastewater treatment.

Proposing a method for online permeability monitoring in membrane bioreactors.

Reliable and efficient operation of membrane bioreactors (MBR) for centralized municipal wastewater treatment strongly depends on a good monitoring of the membrane permeability, a value often used to quantify the transmissibility (i.e. the inverse of the flow resistance) of the membrane. By directly or indirectly evaluating this parameter the operator normally plans on the necessity and effectivity of maintenance activities. To allow the operator monitoring the membrane performance closely, the present paper proposes a method for online permeability based on the measurements of permeate flow, transmembrane pressure and water temperature. The theoretical background of the method is discussed and a data set of 250 days of continuous pilot operation of a municipal MBR equipped with three different standard membrane modules is used for testing. The method is numerically simple enough to allow being implemented on any programmable logical controller.

Urban and rural risks of Lyme disease in the Scottish Highlands.

BACKGROUND: This paper investigates the pattern of Lyme disease testing and infection within the Highland region of Scotland. METHODS: Data from all Highland samples tested during 2004-2006 were analysed according to result and patient's residence in relation to the eight fold Scottish Executive's urban/rural classification, and distance from woodland. RESULTS: In total, 1602 patients were tested for Lyme disease, 0.71% of the Highland population. From these, 104 (6.5%) were seropositive. There were more patients tested, and seropositive patients from rural than urban locations, 1113 vs 489, and 79 vs 25 respectively. There were also significantly more seropositive patients per patients tested from rural locations (chi2, p<0.0001). The number of patients tested and seropositive patients increased as the rural areas become more remote. The likelihood of being tested for Lyme disease also increased as the distance between a patient's residence and woodland decreased. The relative risk of being tested elevated by 74% for those patients living within 200 metres of woodland. CONCLUSIONS: Those living in the most rural areas of Highland and those living closest to woodland have an increased risk of being tested and having Lyme disease.

Agriculture versus wastewater pollution as drivers of macroinvertebrate community structure in streams.

Water pollution is ubiquitous globally, yet how the effects of pollutants propagate through natural ecosystems remains poorly understood. This is because the interactive effects of multiple stressors are generally hard to predict. Agriculture and municipal wastewater treatment plants (WWTPs) are often major sources of contaminants for streams, but their relative importance and the role of different pollutants (e.g. nutrients or pesticides) are largely unknown. Using a 'real world experiment' with sampling locations up- and downstream of WWTPs, we studied how effluent discharges affected water quality and macroinvertebrate communities in 23 Swiss streams across a broad land-use gradient. Variation partitioning of community composition revealed that overall water quality explained approximately 30% of community variability, whereby nutrients and pesticides each independently explained 10% and 2%, respectively. Excluding oligochaetes (which were highly abundant downstream of the WWTPs) from the analyses, resulted in a relatively stronger influence (3%) of pesticides on the macroinvertebrate community composition, whereas nutrients had no influence. Generally, the macroinvertebrate community composition downstream of the WWTPs strongly reflected the upstream conditions, likely due to a combination of efficient treatment processes, environmental filtering and organismal dispersal. Wastewater impacts were most prominently by the Saprobic index, whereas the SPEAR index (a trait-based macroinvertebrate metrics reflecting sensitivity to pesticides) revealed a strong impact of arable cropping but only a weak impact of wastewater. Overall, our results indicate that agriculture can have a stronger impact on headwater stream macroinvertebrate communities than discharges from WWTP. Yet, effects of wastewater-born micropollutants were clearly quantifiable among all other influence factors. Improving our ability to further quantify the impacts of micropollutants requires highly-resolved water quality and taxonomic data with adequate spatial and temporal sampling. These improvements would help to better account for the underlying causal pathways that drive observed biological responses, such as episodic contaminant peaks and dispersal-related processes.

Soft tissue dehiscence coverage around endosseous implants: a prospective cohort study.

AIM: To evaluate the healing outcome of soft tissue dehiscence coverage at implant sites. MATERIAL AND METHODS: Ten patients with one mucosal recession defect at an implant site and a contralateral unrestored clinical crown without recession were recruited. The soft tissue recessions were surgically covered using a coronally advanced flap in combination with a free connective tissue graft. Healing was studied at 1, 3 and 6 months post-operatively. RESULTS: Soft tissue dehiscences were covered with a coronal overcompensation of the flap margin up to 1.2 mm after the procedure. After 1 month, the coverage shrank to a mean of 75%, after 3 months to 70% and after 6 months to 66%. CONCLUSIONS: The implant sites revealed a substantial, clinically significant improvement following coronal mucosal displacement in combination with connective tissue grafting, but in none of the sites, a could complete implant soft tissue dehiscence coverage be achieved.

Are we about to upgrade wastewater treatment for removing organic micropollutants?

Activated sludge treatment allows only for a partial removal of micropollutants, mainly via sorption and biological degradation. Ozonation and activated carbon filtration are processes bearing the potential to drastically reduce the micropollutant load discharged to the environment after (centralized) biological treatment. The estimated total costs between 0.05 and 0.20 euro per m3 treated water (depending on plant size and effluent DOC content) represent only a small fraction of the total costs for urban wastewater management and are therefore considered feasible. Full scale testing is currently planned or under way with the aim to a) confirm this cost estimation and b) to demonstrate the benefit by quantification of the effect of removal and by documenting the impact on the ecology of receiving waters. Ozonation would have the additional advantage of achieving partial disinfection. Another issue currently being intensively studied is the byproducts formed during ozonation and their toxicity. Evidence is needed that the formed ozonation byproducts are either harmless or easily degradable. Since a 5% to 20% loss of sewage is occurring due to sewer leakage and combined sewer overflow an improved reduction of micropollutant input to the aquatic environment requires that advanced centralized treatment is complemented with measures taken before discharge into the sewer. Options hereto may be waste design, labeling of compounds according to environmental friendliness or separate treatment of quantitatively significant point sources (e.g. hospital wastewater, nursery homes, industrial wastewater).

Anammox brings WWTP closer to energy autarky due to increased biogas production and reduced aeration energy for N-removal.

Fifty years ago when only BOD was removed at municipal WWTPs primary clarifiers were designed with 2-3 hours hydraulic retention time (HRT). This changed with the introduction of nitrogen removal in activated sludge treatment that needed more BOD for denitrification. The HRT of primary clarification was reduced to less than one hour for dry weather flow with the consequence that secondary sludge had to be separately thickened and biogas production was reduced. Only recently the ammonia rich digester liquid (15-20% of the inlet ammonia load) could be treated with the very economic autotrophic nitritation/anammox process requiring half of the aeration energy and no organic carbon source compared to nitrification and heterotrophic denitrification. With the introduction of this new innovative digester liquid treatment the situation reverts, allowing us to increase HRT of the primary clarifier to improve biogas production and reduce aeration energy for BOD removal and nitrification at similar overall N-removal.

The use of local isolates in Western blots improves serological diagnosis of Lyme disease in Scotland.

Nine Scottish Borrelia burgdorferi isolates were investigated in IgG Western blot tests. Sera previously found to be positive and negative when tested by routine Western blots prepared from reference strain B. burgdorferi sensu stricto antigen had different outcomes with these isolates. Two isolates, E5 (Borrelia afzelii) and G4 (B. burgdorferi sensu stricto) performed well, reproducing Western blot-positive results in 90 and 95% of tests, respectively. When antigens from both isolates were incorporated into a single IgG Western blot, the results of a panel of sera were improved when compared to the routine reference strain IgG Western blot. All of the sera positive by the routine Western blot remained positive using the Scottish isolate antigen mix. Twenty-three of the 25 negative sera remained negative and two produced an equivocal result. Of the 15 samples that tested IgG Western blot equivocal with the B. burgdorferi sensu stricto reference strain, 11 (73%) became weak or strong positive when tested with the B. afzelii/B. burgdorferi sensu stricto antigen mix (chi(2)=14.35, Yates' correction, P<0.001). In seven of these, a clinical picture of Lyme disease was consistent with the new results. The use of Scottish strains of B. afzelii and B. burgdorferi sensu stricto to provide antigen for the IgG Western blot improves the diagnosis of Lyme disease for patients in Scotland.

Treatment of toilet wastewater for reuse in a membrane bioreactor.

Toilet wastewater is treated and reused on site at Europe's highest membrane bioreactor (MBR), located in a cable car mountain station in the ski resort of Zermatt. Negative impacts on the sensitive mountain environment are minimised by reusing close to 100% of the treated wastewater for toilet flushing. Besides 100% nitrogen removal, 80% of phosphorus was also eliminated. This paper presents operational results, optimisations of sludge management, decoloration and long-term maintenance of biomass in the very low-loaded summer season. From a global view the experiences and results of the project are of great importance, proposing a solution to a problem existing 100-fold in the Alps as well as in arid regions all over the world: reducing water consumption for sanitation by reuse.

Comparison of analytical methods for the evaluation of antibody responses against epitopes of polymorphic protein antigens.

Surface exposed protein antigens of the malaria parasite Plasmodium falciparum frequently harbor multiple dimorphic amino acid positions. These are associated with parasite immune evasion and represent a major obstacle for subunit vaccine design. Here, we have analyzed the flexibility of the humoral immune response against a semiconserved sequence (YX(44)LFX(47)KEKMX(52)L) of the key malaria blood stage vaccine candidate merozoite surface protein-1 (MSP-1). Monoclonal antibodies (mAbs) raised against one of the six described natural sequence variants of MSP-1(43-53) were analyzed for cross-reactivity with the other allelic forms, which differ in one to three positions from the immunizing sequence. Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy demonstrated marked differences in mAb binding avidity to the variant sequences and isothermal titration calorimetry (ITC) provided evidence for a very low affinity of some of the interactions. In immunofluorescence analysis (IFA) and Western blotting analysis, the mAbs nevertheless stained all analyzed parasite clones expressing MSP-1(43-53) variant sequences. When used for the evaluation of humoral immune responses in clinical malaria vaccine trials, these two commonly used methods may thus not be suitable to distinguish biologically functional high affinity antibody responses from irrelevant low-affinity cross-reactivities.

Generation of chimeric monoclonal antibodies from mice that carry human immunoglobulin Cgamma1 heavy of Ckappa light chain gene segments.

Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cgamma2a gene segment (mCgamma2a) with the human Cgamma1 gene segment (hCgamma1), and (ii) the mouse immunoglobulin light chain (IgL) Ckappa gene segment (mC kappa) with its human counterpart (hC kappa). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric kappa or mouse lambda light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCgamma1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCgamma1 and hCkappa gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCgamma1/IgLhCkappa double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCgamma1 associated functions.

Pneumocystis carinii antibody testing.

Sera from blood donors and patients from all over Scotland were examined by indirect immunofluorescence using Pneumocystis carinii antigen from infected rat lung. Antibody was found in 76 of 488 (15.6%) of patients tested on clinical grounds but in only 13 of 148 (8.8%) blood donors. The antibody rates were higher in disease groups likely to have or develop P carinii pneumonia: in those with histologically confirmed or strongly suspected P carinii pneumonia the rate was 14 of 24 (58.3%); in those who had undergone transplantation eight of 24 (33.3%); in those who were immunosuppressed five of 16 (31.2%); in those who were human immunodeficiency virus antibody (HIV) positive 11 of 43 (25.6%); in those with malignancy 34 of 233 (14.6%); and in those with chest infection 10 of 85 (11.7%). P carinii pneumonia was confirmed or likely in four of 45 (8.8%) patients with titres of 1/8-1/16 and in three of seven (42.8%) in those with titres of greater than or equal to 1/128. Seroconversion or rising titre was detected in seven of 13 (53.8%) cases of confirmed or likely P carinii pneumonia compared with 10 of 93 (10.7%) in other patients. Diagnosis of P carinii infection can therefore be assisted by positive immunofluorescence results, but negative serology does not exclude infection.

Toxoplasma dye test using cell culture derived tachyzoites.

AIMS: To assess the diagnostic usefulness of Toxoplasma gondii tachyzoites produced by serial passage in HeLa cell culture. METHODS: Tachyzoites derived from serial passage in cell culture were used in the dye test. Human sera were also examined to determine their suitability for use as accessory factor. Using the optimum conditions, the dye test using cell culture derived tachyzoites was compared with the current method of production (animal culture) on 105 randomly selected sera. Start up and maintenance costs of each system were compared. RESULTS: Tachyzoites in most cell culture harvests (84%) from both early and later passes were useable. Tachyzoite yield and viability were maintained during serial passage in cell culture. Sodium citrate was used to modify accessory factor and improve its suitability. The performance of the accessory factor was improved by the addition of 1% and 3% sodium citrate for the current and cell culture systems, respectively. Under optimum conditions, dye test titres using cell culture and current systems were compared on 105 randomly selected sera. The results from 92 of 105 (87.6%) patients agreed or were within one dilution, but all discrepancies were resolved on re-testing. Start up costs for the current system would be 2.5 times and overall maintenance three times more expensive than cell culture. CONCLUSIONS: Tachyzoites derived from cell culture can be used routinely in the dye test. Production in cell culture is more cost effective than animal culture. It is possible for general hospitals to perform the dye test, thus obtaining faster and more specific results.

Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis.

AIMS: To develop an immunosorbent agglutination assay for the detection of Toxoplasma gondii IgE antibodies (IgE-ISAGA); to assess its specificity; and to determine the role of specific IgE in the diagnosis of current toxoplasma infection. METHODS: Rabbit antihuman IgE capture antibody was adsorbed onto microtitre plates and formaldehyde fixed tachyzoites were used to identify specific antibody. Specificity was assessed in 513 serum samples (blood donor, potentially interfering and difficult, elevated and low total IgE and myeloma). Serum samples (n = 108) from 65 patients with documented toxoplasmosis were tested, as were 26 serum samples from nine pregnant women positive for specific IgM and 30 from 20 HIV positive patients. RESULTS: IgE-ISAGA was highly specific with only three of 513 (0.58%) positive reactions amongst the control groups, one of which (0.19%) was regarded as a false positive. Elevated total IgE did not influence specific IgE results nor did the presence of abnormal immunoglobulin concentrations. Sixty (92.3%) patients with toxoplasma associated lymphadenopathy had specific IgE in one or more samples. Positive or borderline results were obtained in 68 of 77 (88.3%) serum samples taken up to four months after onset and were also detected for up to 11 months in 21 of 31 (67.7%) sera. Of the nine pregnant women with detectable specific IgM, specific IgE was absent in five (12 specimens). Specific IgE was also detected in 10 of 30 (33.3%) serum samples from the 20 HIV positive patients, which was similar to the number with specific IgM. Neither the specific IgE nor IgM tests could distinguish symptomatic from asymptomatic HIV positive patients. CONCLUSIONS: IgE-ISAGA is sensitive, specific, and easy to perform. Although results suggest that specific IgE may be less helpful than previously claimed, specific IgE has a useful role in the diagnosis of current toxoplasma infection when used in conjunction with other tests.

Do IgA, IgE, and IgG avidity tests have any value in the diagnosis of toxoplasma infection in pregnancy?

AIM: To determine the value of tests for specific IgA, IgE, and IgG avidity in diagnosing Toxoplasma gondii infection during pregnancy. METHODS: In a retrospective study, current serological tests (dye test and three IgM assays with different sensitivities) were compared with immunosorbent agglutination assays (ISAGA) for specific IgA and IgE and an IgG avidity enzyme linked immunosorbent assay (ELISA). Patient group 1 comprised six women with definite or probable infection during pregnancy determined by congenital toxoplasmosis or laboratory results. Group 2 comprised seven women infected during or before 11 pregnancies (two consecutive pregnancies in two patients and three in a third). RESULTS: One patient in group 1 seroconverted during pregnancy. IgA ISAGA and avidity confirmed acute infection when confirmatory IgM ELISA remained negative. In five of six patients from group 1, IgA and IgE ISAGA and avidity confirmed acute infection. In group 2, the dye test titre was raised in seven of 11 pregnancies (six of seven patients). Specific IgM and IgA were positive during all 11 pregnancies. IgE ISAGA was positive in only four of 11 pregnancies (three of seven patients), but negative results in the remainder may exclude acute infection. High avidity antibodies indicative of past infection were found in four of 11 pregnancies (two of seven patients). CONCLUSIONS: Each test improved diagnosis or timing of infection but no single test was ideal. The IgA ISAGA was sensitive and detected seroconversion. Positive IgE ISAGA and low avidity both confirmed infection, whereas negative IgE may exclude acute infection. High avidity diagnosed past infection but persistence of low avidity reduced its value to differentiate acute and past infection. Further studies with larger patient groups are needed to determine the optimum diagnostic strategy. These techniques are valuable in complementing existing tests.

Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage?

AIM: To examine three lineages of Toxoplasma gondii RH strain in terms of performance in the dye test, culture, and gene expression. METHODS: Historical data (culture growth and performance in the dye test) from three lineages of RH strain tachyzoites (B, J, and Q) that had been continuously cultured in HeLa cells was assessed. Tachyzoite harvests obtained during continuous cell culture were retrieved from liquid nitrogen and cultured in HeLa cells, providing mRNA that was extracted and used to study gene expression using random amplified polymorphic DNA analysis at different stages of lineage adaptation to continuous culture. RESULTS: The B and Q lineages consistently produced tachyzoites that were successfully used in the dye test and their gene expression was stable after multiple passages. The J lineage had unpredictable growth, tachyzoites unsuitable for use in the dye test, and changing gene expression with multiple passage. CONCLUSION: This study has explained some anomalies in the performance of different stocks of T gondii, and suggests that lineages that are still evolving in cell culture should be avoided.