Identification of Naegleria fowleri in domestic water sources by nested PCR

Abstract

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.

FIG. 1.

Nested-PCR assay of domestic water samples after culture at 37°C for 4 days. Samples were maintained in tissue culture flasks for 4 days at 37°C. Cultures were harvested with a cell scraper and centrifuged to obtain a pellet and supernatant. The pellet was used for nested PCR, and the supernatant was discarded. The first and second PCR-positive and -negative controls were examined on the gel since this was the first PCR assay performed with domestic water samples. (A) Nested-PCR products were demonstrated on a 4% NuSieve gel. Lanes: a and j, 100-bp ladder; b, positive control for first PCR; c, negative control for first PCR; d, positive control for second PCR; e, negative control for second PCR; f, sample 2; g, sample 3; h, sample 4; and i, sample 5. (B) Nested PCR products were demonstrated on a 1.5% agarose gel. Lanes: a and m, 100-bp ladder; b, positive control for first PCR; c, negative control for first PCR; d, positive control for second PCR; e, negative control for second PCR; f, sample 11; g, sample 12; h, sample 13; i, sample 14; j, sample 15; k, sample 16; l, sample 17. Asterisks indicate samples that were positive for N. fowleri by PCR.

FIG. 2.

Samples cultured for 1 week (A) and 1 month (B) at 37°C were analyzed by PCR. The samples in tissue culture flasks were harvested with a sterile cell scraper and centrifuged to obtain a supernatant and pellet. The pellet was used for nested PCR. PCR products were demonstrated on a 4% NuSieve gel. (A) Lanes: a and o, 100-bp ladder; b, positive control; c, negative control; d, sample 11; e, sample 12; f, sample 13; g, sample 14; h, sample 15; i, sample 16; j, sample 17; k, sample 18; l, sample 19; m, sample 20; n, sample 23. (B) Samples for PCR were prepared after 1 month in continuous culture. Lanes: a, 20-bp ladder; b, positive control; c, negative control; d, sample 2; e, sample 3; f, sample 4; g, sample 6; h, sample 11; i, sample 12; j, sample 14; k, sample 15; l, sample 16; m, sample 17; n sample 20; o, sample Wynd II filter. All domestic samples tested after 1 month were positive, with the exception of sample 20. Asterisks indicate samples that were positive by PCR analysis.

FIG. 3.

Western immunoblot analysis of domestic water samples by using polyclonal anti-N. fowleri and anti-Acanthamoeba antibodies and monoclonal antibody 5D12 for N. fowleri. Samples harvested at 1 month of culture at 37°C were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and separated proteins were transferred to nitrocellulose membranes. The membranes were incubated in the primary antibodies rabbit polyclonal anti-N. fowleri (A), monoclonal 5D12 anti-N. fowleri (B), and rabbit polyclonal anti-Acanthamoeba (C). Secondary antibodies were goat anti-rabbit or rabbit anti-mouse antibodies. The blots were developed by using enhanced chemiluminescence. Lane Nf, a whole-cell lysate of N. fowleri (ATCC 30894) used as a control. Other lanes are labeled with the sample numbers identified in Table 1.

FIG. 4.

Cultures of domestic samples were observed daily for the presence of trophozoites, cysts, or flagellates. (A) Limax-type amoebae were observed in sample 16, which was positive for N. fowleri by PCR. (B) Acanthamoeba cysts were observed in sample 7, which was negative for N. fowleri by PCR. Magnification, ×400.