Comparison of a nested polymerase chain reaction--restriction fragment length polymorphism method, the PATH antigen detection method, and microscopy for the detection and identification of malaria parasites.

A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, the PATH antigen detection method, and light microscopy were compared for their capacity to detect and identify Plasmodium species. One hundred and thirty-six blood specimens obtained from patients suspected of having malaria were examined by each of the three methods. Forty-four specimens were positive for malaria using microscopy as the "gold standard". The sensitivity for nested PCR was 100%, and the specificity was 98%. For the detection of Plasmodium falciparum, the antigen detection method had a sensitivity of 100% and a specificity of 97%. Species identification obtained using PCR-RFLP was identical or superior to light microscopy in 42 cases (96%). Although the nested PCR-RFLP method was more sensitive and specific, the rapid turnaround time and high sensitivity of the antigen detection method makes it a useful adjunct to standard microscopy.

Evidence of delays in transferring patients with methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus to long-term-care facilities.

This retrospective case-control study examined whether there was a difference in length of time awaiting long-term-care placement for patients identified as having methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus compared to controls. Thirty-nine patients with methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus waited for placement an average of 61 days longer than controls (P<.0002). The average number of requests for placement was 2.5 compared to 1.7 for controls (P=.015).

Evaluation of molecular techniques to biotype Giardia duodenalis collected during an outbreak.

Twenty-seven Giardia duodenalis cyst-positive specimens (human, animal, or drinking water) were obtained from a waterborne outbreak in a community in British Columbia, western Canada. Parasite isolates were characterized using molecular techniques at 4 different steps of organism retrieval. None of the drinking water samples (n = 20) infected gerbils and none was successfully amplified using polymerase chain reaction (PCR). We were able to genotype 4 of 7 (human and animal) isolates by amplification of DNA from original specimens at the triosephosphate isomerase (tpi) gene locus using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Five of the original specimens inoculated into Mongolian gerbils (Meriones unguiculatus) were infective and genotyped at the tpi locus using parasite material collected from the gerbil (cysts and trophozoites). Pulsed field gel electrophoresis (PFGE) was used to biotype trophozoites collected from the gerbils as well as trophozoites from the 4 isolates that adapted to culture. Four of these 5 isolates displayed the same (designated outbreak) biotype at all parasite retrieval steps with all molecular techniques including the originally amplified isolates. PCR-RFLP identified an additional biotype group. The 4 isolates that adapted to in vitro culture were also characterized by isoenzyme electrophoresis (IE). Biotype groups identified in these axenized isolates were all the same with each molecular technique (PCR-RFLP, PFGE, IE) tested. Results of this study demonstrate a need for more sensitive molecular methods to detect and characterize Giardia in original host and environmental samples. Results are also consistent with evidence of biotype changes that occur during the presently used process of isolate retrieval.

Molecular epidemiology of cryptosporidiosis outbreaks and transmission in British Columbia, Canada.

Isolates from 25 (13 sporadic and 12 outbreak) cryptosporidiosis cases, 24 of which were from British Columbia, Canada, were characterized using nested polymerase chain reaction amplification of the polymorphic internal transcribed spacer 1 locus. Two predominant Cryptosporidium parvum genotypes were found. Twelve (8 sporadic and 4 outbreak) isolates amplified with the cry7/cry21 primer pair and 12 (5 sporadic and 7 outbreak) isolates amplified with the cry7/cryITS1 primer pair. Multi-locus gene analysis using sequence polymorphisms on 3 other loci, i.e., the thrombospondin-related adhesion protein gene, the dihydrofolate reductase gene, and the 18S rRNA gene on 8 (4 outbreak and 4 sporadic) isolates showed non-random association among the human and animal alleles of the 4 different C. parvum gene loci. Associations between these 2 parasite genotypes and different routes of cryptosporidiosis transmission such as zoonotic, anthroponotic, and waterborne transmission were studied using municipal population and agricultural information, as well as detection of C. parvum oocysts in municipal drinking water specimens of the residential communities of sporadic and outbreak cases.

The toilet as a transmission vector of vancomycin-resistant enterococci.

An elderly woman, admitted to the intensive care unit of a large university teaching hospital, was found to be colonized with vancomycin-resistant enterococci leading to the temporary closure of the unit. She had acquired the organism nosocomially, most likely from an environmental source, which had been contaminated when the toilet of a former patient, also colonized with vancomycin-resistant enterococci, had become blocked and overflowed throughout his and the adjoining room. This is the first report of a hospital toilet as the transmission vector for vancomycin-resistant enterococci.

Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia.

Infection of suckling mice with Giardia trophozoites recovered from the intestines of 11 dogs autopsied in Central and Southern Australia in each case produced an established isolate. In contrast, only 1 isolate was obtained by inoculation of faecal cysts. The organisms grew poorly in comparison with isolates from humans or non-canine animal hosts. Light microscopy revealed that the trophozoites had median bodies with the 'claw hammer' appearance typical of G. intestinalis (syn. G. duodenalis, G. lamblia) but that they differed in shape and nuclear morphology from axenic isolates of human or canine origin. Allozymic analysis of electrophoretic data representing 26 loci and phylogenetic analysis of nucleotide sequences obtained from DNA amplified from the glutamate dehydrogenase locus showed that the 11 isolates examined from Australian dogs were genetically distinct from all isolates of G. intestinalis that have been established previously from humans and animals, and also from G. muris. Both analytical methods placed 10 of the Australian canine isolates into a unique genetic lineage (designated Assemblage C) and the eleventh into a deep-rooted second branch (designated Assemblage D), each well separated from the 2 lineages (Assemblages A and B) of G. intestinalis that encompass all the genotypes known to infect humans. In contrast, 4 axenic isolates derived from dogs in Canada and Europe (the only other isolates to have been established from dogs) have genotypes characteristic of genetic Assemblages A or B. The findings indicate that the novel Giardia identified in these rural Australian dogs have a restricted host range, possibly confined to canine species. The poor success rate in establishing Giardia from dogs in vitro suggests, further, that similar genotypes may predominate as canine parasites world-wide. The absence of such organisms among isolates of Giardia that have been established from humans by propagation in suckling mice indicates that they are unlikely to infect humans. However, infection of humans by those dog-derived genotypes that grow in vitro cannot be excluded.

Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma Investigation Team.

BACKGROUND: Outbreaks of toxoplasmosis are recognised infrequently. In March, 1995, a sudden increase of serologically diagnosed cases of acute toxoplasmosis was noted in the Greater Victoria area of British Columbia, Canada. Concurrently, but independently, seven cases of acute toxoplasma retinitis were diagnosed against a background of no cases in the previous 5 years. METHODS: Cases were defined by serological testing, clinical presentation, and residence in Greater Victoria. A screening programme for women who were or had been pregnant was started. Geographical mapping of cases, and case-control studies of symptomatic cases and of women enrolled in the screening programme were done. FINDINGS: 100 individuals aged 6 to 83 years met the definition for an acute, outbreak-related case. 94 resided in Greater Victoria and six had visited it; 19 had retinitis, 51 had lymphadenopathy, four others had symptoms consistent with toxoplasmosis, seven had other symptoms, 18 were symptom-free, and one would not provide information. 36 (0.9%) of 3812 screened pregnant and postnatal women were cases. Excess cases were not detected outside Greater Victoria and no conventional source of toxoplasmosis was implicated. Mapping studies of cases and of the screened women, and both case-control studies showed significant associations between acute infection and residence in the distribution system of one reservoir supplying water to Greater Victoria (ORs or RRs: 3.53, 3.05, 8.27, and 5.42, respectively). The epidemic curve appeared bimodal, with peaks in December, 1994, and March, 1995, that were preceded by increased rainfall and turbidity in the implicated reservoir. INTERPRETATION: A municipal water system that uses unfiltered, chloraminated surface water was the likely source of this large community-wide outbreak of toxoplasmosis.

False-positive results in immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test.

Although tests for detection of immunoglobulin M (IgM) toxoplasma antibodies have been reported to have a high degree of accuracy, it is well recognized by investigators in the United States and Europe that false-positive results may occur with many of these tests, at times to an alarming degree. Unfortunately, this information is not well documented in the literature. Studies on various toxoplasma IgM test kits are frequently flawed. The investigators often use reference tests which have not previously been carefully evaluated as well as sera that were not appropriate to answer the question of how often false-positive results might occur. We recently had the unique opportunity to evaluate the accuracy of the Platelia Toxo IgM test in 575 serum samples obtained during an outbreak of toxoplasmosis which occurred in 1995 in the Capital Regional District of British Columbia, Canada. When compared with results obtained in a reference IgM enzyme-linked immunosorbent assay (ELISA), the Platelia Toxo IgM test had a sensitivity of 99.4%, specificity of 49.2%, positive predictive value of 51.9%, negative predictive value of 99.3%, and an overall agreement of 67.0%. In an attempt to resolve discrepancies between these two tests, a serological profile (Sabin-Feldman dye test, IgA and IgE antibody tests, differential agglutination [AC/HS] test, and IgG avidity method) was performed. Of 153 serum samples that were positive in the Platelia Toxo IgM test and negative in the IgM ELISA, 71 (46.4%) were negative in the Sabin-Feldman dye test. Of the serum samples that were positive in the dye test, 77 (93.9%) had a serological profile most compatible with an infection acquired in the distant past. These results reveal high numbers of false-positive results in the Platelia Toxo IgM test and highlight the importance of appropriate evaluation of commercial tests that are currently being marked. Our results also emphasize the importance of confirmatory testing to determine whether the results of an IgM antibody test reflect the likelihood of a recently acquired infection.

Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada.

This study was carried out to estimate the prevalence and potential for human infectivity of Giardia cysts in Canadian drinking water supplies. The presence of Cryptosporidium oocysts was also noted, but isolates were not collected for further study. A total of 1,760 raw water samples, treated water samples, and raw sewage samples were collected from 72 municipalities across Canada for analysis, 58 of which treat their water by chlorination alone. Giardia cysts were found in 73% of raw sewage samples, 21% of raw water samples, and 18.2% of treated water samples. There was a trend to higher concentration and more frequent incidence of Giardia cysts in the spring and fall, but positive samples were found in all seasons. Cryptosporidium oocysts were found in 6.1% of raw sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples. Giardia cyst viability was assessed by infecting Mongolian gerbils (Meriones unguiculatus) and by use of a modified propidium iodide dye exclusion test, and the results were not always in agreement. No Cryptosporidium isolates were recovered from gerbils, but 8 of 276 (3%) water samples and 19 of 113 (17%) sewage samples resulted in positive Giardia infections. Most of the water samples contained a low number of cysts, and 12 Giardia isolates were successfully recovered from gerbils and cultured. Biotyping of these isolates by isoenzyme analysis and karyotyping by pulsed-field gel electrophoresis separated the isolates into the same three discrete groups. Karyotyping revealed four or five chromosomal bands ranging in size from 0.9 to 2 Mb, and four of the isolates had the same banding pattern as that of the WB strain. Analysis of the nucleotide sequences of the 16S DNA coding for rRNA divided the isolates into two distinct groups corresponding to the Polish and Belgian designations found by other investigators. The occurrence of these biotypes and karyotypes appeared to be random and was not related to geographic or other factors (e.g., different types were found in both drinking water and sewage from the same community). Biotyping and karyotyping showed that isolates from this study were genetically and biochemically similar to those found elsewhere, including well-described human source strains such as WB. We conclude that potentially human-infective Giardia cysts are commonly found in raw surface waters and sewage in Canada, although cyst viability is frequently low. Cryptosporidium oocysts are less common in Canada. An action level of three to five Giardia cysts per 100 liters in treated drinking water is proposed on the basis of the monitoring data from outbreak situations. This action level is lower than that proposed by Haas and Rose (C. N. Haas and J. B. Rose, J. Am. Water Works Assoc. 87(9):81-84, 1995) for Cryptosporidium spp. (10 to 30 oocysts per 100 liters).

A second community outbreak of waterborne giardiasis in Canada and serological investigation of patients.

A waterborne outbreak of giardiasis which occurred 5 years after another in the same town in Canada was investigated. Sera from residents defined as cases or non-cases were tested by enzyme-linked immunosorbent assay (ELISA) and compared with sera from symptomatic and asymptomatic control groups. The outbreak-associated Giardia isolate was retrieved from contaminated drinking water and antigen from this strain was used in the serological investigation. Up to 84% of cases were identified by ELISA. More cases were identified by elevated immunoglobulin (Ig) G than by either elevated anti-Giardia IgA or IgM levels. Residents of the community infected during the first outbreak were significantly less likely to have been reinfected during the second outbreak. This is the first report of a second waterborne outbreak occurring in a community and results of the investigations are consistent with an acquired, protective immunity lasting at least 5 years.

Characterization of Giardia duodenalis isolates from a waterborne outbreak.

Isolates were retrieved from drinking water and from animal and human sources associated with a waterborne outbreak of giardiasis. This is the first report of water-source and epidemic-associated Giardia isolates being adapted to in vitro propagation. Outbreak-associated, non-out-break-associated, and reference isolates were characterized using isoenzyme electrophoresis and pulsed-field gel electrophoresis (PFGE). All outbreak-associated and 2 other isolates were in one of eight zymodemes. The chromosomal complement of the outbreak-associated isolates was relatively homogeneous; this PFGE karyotype was distinguishable from other karyotypes. Overall results of both characterization methods were similar, although PFGE appears to be a more discriminating biotyping technique. Banding patterns of the outbreak-associated Giardia isolates remained the same even though the parasite passed through different hosts during the outbreak. Heterogeneity of isolates was also demonstrated for the first time within a single community not associated with the outbreak.

Comparison of an in vitro method and an in vivo method of Giardia excystation.

An in vitro method and an in vivo method of excystation were compared to determine the most useful method for the retrieval of Giardia duodenalis isolates. Cysts from 11 Giardia strains were used. In vitro excystation produced motile trophozoites in 16 sets, while in vivo excystation produced trophozoites in all of the 21 comparative sets of excystations. Few cultures were lost because of contamination by either method (17% of in vitro-derived trophozoites versus 23% of in vivo-derived trophozoites; P greater than 0.05). Both methods demonstrated comparable isolate retrieval rates (15% of in vitro-derived trophozoites adapting to culture compared with 29% of in vivo-derived trophozoites; P greater than 0.05), although analysis of the strains retrieved showed that two isolates were retrieved from in vitro excystation alone, compared with four from in vivo excystation. Analysis that included results of extra in vivo cultures showed that a total of nine isolates were retrieved by using this type of excystation. Despite the disadvantages of cost and labor, in vivo excystation appears to be more useful than in vitro excystation for isolate retrieval at the present time.

Factors associated with acquiring giardiasis in British Columbia residents.

Giardia is the most frequently reported intestinal parasite in Canada. More than 2,000 cases are reported annually in British Columbia which exceeds the number of cases of either campylobacteriosis or salmonellosis. Since the different ways this parasite is spread in British Columbia have not been determined, our purpose was to investigate certain factors that might be associated with acquiring giardiasis in this province. Telephone interviews provided information from a group of infected persons and from a group of non-infected control subjects. Information obtained from these interviews was used to identify associated risk factors in this group of cases. If results of the study are generalized to the population-at-risk, they indicate that water (drinking and recreational) is an important vector for transmission of Giardia in British Columbia.

Laboratory diagnosis of giardiasis.

The epidemiology, clinical presentations, and recent developments in understanding Giardia are reviewed. Diagnosis is discussed in light of recent studies that challenge the clinician's approach to the diagnosis of enteric parasites, including giardiasis, and that demonstrate the need for further evaluation on the basis of cost-effectiveness, as well as reliability and clinical practicality. The overall effectiveness and difficulties associated with present standard diagnostic methods and the more recently developed immunologic approaches to diagnosis in giardiasis are reviewed.

Immunological methods of diagnosis in giardiasis: an overview.

The epidemiology and clinical presentations of giardiasis are reviewed to provide a basis of understanding the laboratory aspects of this parasitic infection. Diagnosis is then discussed in light of recent studies that challenge our overall approach to the diagnosis of enteric parasites and demonstrate the need for further evaluation on the basis of cost-effectiveness as well as reliability and clinical practicality. Overall effectiveness as well as the difficulties with present standard diagnostic methods in giardiasis are reviewed. This is followed by a discussion of the more recently developed immunological approaches to diagnosis. The role of these tests in the parasitology laboratory is also discussed.

Comparison of in vitro antimicrobial susceptibilities of Mycobacterium avium-M. intracellulare strains from patients with acquired immunodeficiency syndrome (AIDS), patients without AIDS, and animal sources.

Difloxacin, A-56620, cefazolin, cefotaxime, ceftizoxime, cephapirin, SK&F 88070, and spectinomycin were used to compare the in vitro susceptibilities of Mycobacterium avium-M. intracellular isolates from patients with acquired immunodeficiency syndrome (AIDS), patients without AIDS, and diseased animals. Against the isolates from humans without AIDS, the quinolone compounds difloxacin and A-56620 were found to be the most effective, each inhibiting 50% of strains at a concentration of 2 micrograms/ml. The remaining antimicrobial agents had MICs for 50% of strains tested of at least 32 micrograms/ml. Statistically significant differences were observed in the antibiogram patterns among the M. avium-M. intracellulare strains from each of the three sources.

Isoenzyme analysis of human and animal isolates of Giardia duodenalis from British Columbia, Canada.

Isoenzyme patterns of 32 isolates of Giardia duodenalis, obtained from 6 beavers and 11 humans from British Columbia, plus 15 other isolates were evaluated using thin-layer starch-gel electrophoresis. We attempted to use 12 enzymes; 9 gave reproducible and interpretable results. The isoenzyme patterns of the isolates were classified into 12 groups with 17 (53%) of the 32 isolates confined to 1 group. The other 11 groups each comprised only 1 or 2 isolates. There was no obvious correlation between clinical symptoms and isoenzyme patterns. Our findings suggest that beavers, like humans and gerbils are receptive to organisms with many different isoenzyme patterns.

Isoelectric focusing of ten strains of Giardia duodenalis.

Ten strains of human- and animal-source Giardia duodenalis were evaluated using an isoelectric focusing technique. Banding patterns obtained from total cell proteins of trophozoites demonstrated both similarities and differences between strains. This confirms the heterogeneity of this morphological group of Giardia sp. demonstrated by others. Heterogeneity was demonstrated among the strains retrieved from human and animal hosts and from hosts within the same geographical region.

Giardia duodenalis: enhanced growth in cell culture.

Growth of Giardia duodenalis in broth and in animals has been studied in considerable detail. In contrast, the kinetics of growth in cell culture have been little evaluated. In this study, in vitro growth of G. duodenalis was evaluated in cell culture, primarily using mouse McCoy cells in vials. The media used were Giardia broth (TYI-G), Trichomonas vaginalis broth (TYI-T), and standard cell culture media (CMGA) alone and in combination (2 parts by volume CMGA to one part of TYI broth). Addition of cell culture enhanced the sensitivity of the systems in detecting low numbers of G. duodenalis. Growth was identified consistently with inocula less than or equal to 10/ml, and often with a calculated 10-1/ml inoculum with CMGA/TYI-T and CMGA/TYI-G with cells, and with TYI-G with and without cells. The 2 preferred systems for sensitivity and growth were CMGA/TYI-G with cells and TYI-G with cells. The pH fell minimally in the growth systems and, if CMGA was in the media, cell monolayers remained intact and viable throughout the experiment. In preliminary experiments, cell cultures did not allow growth of one strain of G. muris. These cell culture systems may be useful for detection of low numbers of non-laboratory adapted trophozoites, and should be useful in evaluating the interaction of G. dudodenalis with cells in culture.

Inner-city survey for tuberculosis: evaluation of diagnostic methods.

A total of 1,271 persons living in a socially and economically depressed, inner-city area of Vancouver, British Columbia, voluntarily attended a tuberculosis case-finding campaign. Chest x-ray, on-the-spot specimen of sputum, and tuberculin skin test were offered at the time of the first attendance. All 3 diagnostic methods were found to be well accepted, with 93% of the participants having an x-ray, over 95% producing a sputum specimen, and almost 95% having a tuberculin test (a quarter of these did not, however, report for reading of the test). Eight cases of bacteriologically confirmed pulmonary tuberculosis were found: 6 suspected on x-ray (the remaining 2 films were abnormal but not diagnostic of tuberculosis), and 6 being positive on smear and/or culture of the initial on-the-spot sputum specimen. Examination of a second specimen of sputum diagnosed all of the 8 active cases identified by the survey. These results suggest that, in this particular setting, a chest radiogram taken by a transportable chest x-ray apparatus or examination of 2 sputum specimens might be equally successful at detecting all cases of active pulmonary tuberculosis within the time required for sputum culture. Examination of the sputum smear immediately identifies all the more infectious cases of pulmonary tuberculosis. The prevalence rate of 629 per 100,000 among those presenting themselves to this campaign illustrates the high-yield which might be achieved by active case-finding projects in known high-incidence segments of a generally low-incidence population.