In July 1998, the mother of an 18-month-old boy in rural Tennessee found a triatomine bug in his crib, which she saved because it resembled a bug shown on a television program about insects that prey on mammals. The gut contents of the Triatoma sanguisuga were found, by light microscopy and polymerase chain reaction (PCR), to be infected with Trypanosoma cruzi; PCR products hybridized with T. cruzi-specific oligonucleotide probes. Whole-blood specimens obtained from the child in July and August were negative by buffy-coat examination and hemoculture but positive by PCR and DNA hybridization, suggesting that he had low-level parasitemia. Specimens obtained after treatment with benznidazole were negative. He did not develop anti-T. cruzi antibody; 19 relatives and neighbors also were seronegative. Two of 3 raccoons trapped in the vicinity had positive hemocultures for T. cruzi. The child's case of T. cruzi infection-the fifth reported US autochthonous case-would have been missed without his mother's attentiveness and the availability of sensitive molecular techniques.

Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological comparisons. Previous serological studies have utilized a variety of antigen preparations and assay methods and reported disparate (3 to 100%) results. This study was designed to (i) optimize an enzyme-linked immunosorbent assay for detecting serum antibodies to each of the Acanthamoeba serogroups and (ii) test 55 healthy individuals for specific immunoglobulin G reactivity. The highest signal-to-background ratio was found when 3,000 fixed, intact trophozoites per well were used with a 1:10 serum dilution. Sera yielding optical densities of <0.25 against all three Acanthamoeba serogroups were used to define the cutoff for positive results. The highest background reactivity with these sera was seen with Acanthamoeba polyphaga (serogroup 2), followed by Acanthamoeba culbertsoni (serogroup 3) and Acanthamoeba astronyxis (serogroup 1). Of 55 subjects tested, the highest number of positive results was seen with A. polyphaga (81.8%), followed by A. astronyxis (52.8%) and A. culbertsoni (40%). Seven serum samples (12.7%) were negative for all three Acanthamoeba serogroups, 16 (29.1%) were positive for one serogroup only, 16 were positive for two serogroups, and 16 reacted to all three serogroups. Further analysis showed no significant associations between serogroup reactivity and age or gender. However, some ethnic differences were noted, especially with A. polyphaga antigens. In that case, serum samples from Hispanic subjects were 14.5 times less likely to be positive (P ‫؍‬ 0.0025) and had lower mean absorbance values (P ‫؍‬ 0.047) than those from Caucasian subjects. Overall, these data suggest that Acanthamoeba colonization or infection is more common than previously thought. Mild or asymptomatic infections may contribute to the observed serum reactivities.

Investigators using light microscopy have identified the protozoan parasite Entamoeba gingivalis from diseased gingival pockets for nearly 100 years. The objective of the present investigation was to develop a molecular biology approach for determining the presence of E. gingivalis in both diseased gingival pockets and healthy gingival sites. For this, a previously developed conventional polymerase chain reaction (PCR) was evaluated and a real-time polymerase chain reaction assay was developed. Paper points were inserted into the base of the sulcus of both diseased gingival pockets and healthy gingival sites. DNA was extracted using the QIAamp DNA mini kit, and subsequently analyzed using conventional and real-time PCR analysis. A previously described primer set specific for the small subunit ribosomal RNA gene (SSU rDNA) of E. gingivalis was used for the conventional PCR. For the real-time PCR, a primer set was designed to amplify a 135-bp fragment inside the SSU rDNA of E. gingivalis. A conventional PCR assay detected E. gingivalis in 27% of diseased gingival pockets. The real-time PCR using a different primer set detected protozoa in 69% of diseased pocket sites. Thus, the latter technique proved more sensitive for detection of E. gingivalis. No E. gingivalis were detected in any of the healthy gingival pocket sites using either type of PCR assay. Results support a concept that the presence of E. gingivalis is associated only with diseased gingival pocket sites. The newly described methodology may also serve to provide a novel eukaryotic cell marker of disease status in gingival pockets.

Using electron microscopy we documented some of the intracellular events that occur in Naegleria fowleri suspended in Page amoeba saline after ingestion of Legionella pneumophila. Photomicrographs showed intracellular vacuoles containing bacteria in the process of binary fission that was accompanied by alignment of mitochondria and ribosome-like structures along the vacuole membrane. Although these intracellular events are remarkably similar to that seen in Legionella replication within human monocytes, we could not demonstrate an increase in the number of bacteria by CFU or dark-field microscopy. However, when the Naegleria cells were allowed to ingest Legionella cells while suspended in amoeba culture medium, the number of bacteria increased, and this was contingent upon the presence of viable amoebae.

There are numerous in vitro studies documenting the multiplication of Legionella species in free-living amoebae and other protozoa. It is believed that protozoa serve as host cells for the intracellular replication of certain Legionella species in a variety of environmental settings. This study describes the isolation and characterization of a bacterium initially observed within an amoeba taken from a soil sample. In the laboratory, the bacterium multiplied within and was highly pathogenic for Acanthamoeba polyphaga. Extracellular multiplication was observed on buffered charcoal yeast extract agar but not on a variety of conventional laboratory media. A 16S rRNA gene analysis placed the bacterium within the genus Legionella. Serological studies indicate that it is distinct from previously described species of the genus. This report also describes methods that should prove useful for the isolation and characterization of additional Legionella-like bacteria from free-living amoebae. In addition, the characterization of bacterial pathogens of amoebae has significant implications for understanding the ecology and identification of other unrecognized bacterial pathogens.