DETECTION AND IDENTIFICATION OF NAEGLERIA FOWLERI REPORT TO THE DEPARTMENT OF THE ENVIRONMENT FINAL PROJECT REPORT MARCH 15 1993 MARCH 31 1995
Report No DWI0708

Feb 1996

SUMMARY

The contract, from March 1993 to March 1995 was funded by the Department of the Environment (reference PECD 7/7/412) and was carried out under the supervision of the Drinking Water Inspectorate.

OBJECTIVES

To develop and test Polymerase Chain Reaction (PCR) and other techniques for the specific identification of Naegleria fowleri from environmental sources. Adaptation of the techniques to enable safe, non-radioactive probes to be used. Development of colorimetric methods for PCR which are semi-quantitative and applicable to large numbers of samples. Studies on optimal isolation of the organism from water and a limited programme of environmental sampling were also envisaged following the development of identification techniques, in order to develop a suitable detection protocol for further testing.

BACKGROUND

N. fowleri is a free -living amoeba found in warm fresh water. It causes a rare and usually fatal, meningitis following infection while swimming. A fatality has occurred, associated with contamination of the hot mineral springs in Bath in 1978 and the organism has recently been detected in power station effluent in the UK.

We can expect N.fowleri colonisation of heated fresh water habitats, such as warm swimming pools and fish farms, with consequent risk of deaths.

Reliable methods for detection and identification of the organism are required. Non-pathogenic species of Naegleria inhabit many waters and may not only be mistaken for the pathogen but also interfere with its isolation.

The knowledge exists to detect microorganisms such as N. fowleri using molecular biological techniques. DNA probes have potential to detect a particular microorganism specifically by virtue of unique sequences of bases in its DNA. PCR enables detection of minute numbers of organisms in water samples or early cultures using DNA amplification and a DNA probing process. The integration of DNA methods into water sampling procedures offers the potential for more specific and sensitive techniques.

STRATEGY

The studies were carried out in the Applied Molecular Biology Unit in the Department of Medical Parasitology, London School of Hygiene and Tropical Medicine. DNA sequences for testing were obtained from the literature and from GenBank. The plan was to design primers and probes based on analysis of available sequences, then to test them for their specificity and sensitivity. Having obtained suitable results, the method would be adapted to use non-radioactive safe probes. Then the development of a colorimetric method capable of high throughput in the detection of specific PCR product would begin. Having optimised this technique, the application to detection of organisms from water samples would be developed, and finally tests on samples from the environment would be carried out. An optimal protocol would then be drawn up for further testing by other groups.

PCR FOR DETECTION OF N FOWLERI

PCR optimisation:

Primers and DNA probes were developed from 2 available gene sequences. Ability of specific PCR methods to detect one organism in laboratory conditions was confirmed. Safe methods of probe labelling using digoxigenin were optimised.

Enumeration:

Currently, cultivation is the only technique able reliably to measure concentrations of the viable organisms. A microtitre plate method of cultivation is better than the present method on Petri plates. Most probable number method can be applied to cultures from water samples after serial dilution.

Identification:

Apart from culturing at temperatures higher than 37C to encourage growth of the thermophilic amoebae N.fowleri, N.lovaniensis and N.australiensis, and avoiding the use of animals, it is best to confine the identity of N. fowleri using isoenzyme, DNA probe or Polymerase Chain Reaction (PCR) methods. We have found that N.fowleri has a lower mobility of glucose phosphate isomerase than other Naegleria spp. A lysate suitable for electrophoretic analysis can be prepared from as few as 5000 organisms.

Using PCR primers for the amoebic mitochondrial ATPase gene we were able to detect 400 organisms but not 40 by fluorescence and size of the PCR product in agarose gel electrophoresis. When a [32P]radiolabelled specific probe was used to detect N. fowleri PCR product, the sensitivity was greatly increased, and DNA from I or fewer organisms was detected. The technique was adapted using the safe label digoxygenin for the probe, and specificity was confirmed in a simple blot-hybridisation protocol.

Using primers for amoebic serine carboxypeptidase we confirmed the specificity of a PCR reported by Sparagano using a safe, digoxygenin-labelled probe.

Development work was carried out on a PCR-Solution Hybridisation Enzyme Linked Assay (PCR-SHELA) which we have used successfully for the dysentery amoeba. PCR-SHELA allows a rapid, highly sensitive semi-quantitative visualisation of PCR product from a large number of reactions without use of hazardous or expensive materials.

Conventional PCR techniques depend on carcinogenic ethidium bromide for staining electropherograms which are examined under ultra violet light, and probing the product with radioactive probes. The method is hazardous, tedious and not easily applicable to large numbers of samples. A colorimetric technique carried out in microtitre 96-well plates, the PCR-Solution Hybridisation Enzyme Linked Assay (PCR-SHELA) would allow a rapid, highly sensitive visualisation of specific PCR product, without use of hazardous or expensive materials. The amplified specific DNA sequence, labelled with biotin, is identified using a digoxigenin-labelled specific internal probe. On addition to a microtitre plate well which has been coated with avidin, DNA strands which are biotinylated are bound and the digoxigenin label can be detected with enzyme-labelled antibody. A reaction linked to the enzyme gives a colour which can be detected visually or using an ELISA reader. The technique is semiquantitative and susceptible to automation for large numbers of samples. A modification of this technique applicable to N. fowleri has been developed by us which not only has the potential for a large throughput and automated reading of the colour reaction, but also has the high sensitivity of a nested PCR technique. In the modified demi-nested PCR-SHELA the two consecutive PCRs of the nested technique are carried out in the same tube which is kept sealed throughout, avoiding cross-contamination which is a problem for other nested reactions. The tube is opened only for the colorimetric detection stage in the microtitre plate, which is not susceptible to contamination.

The technique is specific for the pathogen N.fowleri and will currently detect 10 organisms. Used together with the modified initial concentration and culture protocol we have developed, it will enable N.fowleri to be detected in water supplies even at low concentration and in the presence of other organisms.

After optimisation of the technique S samples kindly supplied by Dr. Simon Kilvington were tested blind, and the 3 N.fowleri samples (from Hong Kong, Bath Spa and an English power station), were detected accurately No detectable colour was seen in the tests on the two other samples, N.australiensis and N. lovaniensis.

PRACTICAL IMPLICATIONS

The PCR protocols we have developed are applicable to specific identification of Naegleria fowleri among amoebae isolated and enumerated in microtitre plate cultures from water samples. Protocols for conventional PCR and for probing the product on membr anes with digoxigenin-labelled probes are tedious and are unlikely to be adopted by the water industry. Direct DNA probing without PCR (not studied here), similarly, is unlikely to be practicable on a large scale except in specialist laboratories. The relatively simple technique of isoenzyme electrophoresis can be applied for identification, but demands large numbers of organisms. The demi-nested PCR-SHELA method, will, we believe, be usable in the water-industry because of its relative simplicity, high throughput and capacity for automation.

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