PCR-ELISA: a new simplified tool for tracing the source of cryptosporidiosis in HIV-positive patients.

Cryptosporidium parvum is a major parasitic cause of death in end-stage AIDS patients that results from both zoonotic and person-to-person transmission. Recent studies have provided evidence that parasites causing zoonotic disease and those causing anthroponotic infection are genetically distinct. Isolates carrying "animal"-type genetic markers were presumed to be the result of zoonotic spread, either directly or through contaminated food and water. The need for a genotype-specific diagnostic tool that can provide clues as to the origin and possible modes of spread of C. parvum strains has been recognised. Here, we report the development of such a tool for C. parvum based on polymerase chain reaction-enzyme linked immunosorbent assay that enables the accurate typing of isolates from HIV-seropositive and HIV-negative patients presenting with diarrhoea from the United Kingdom and Canada. This study also showed that zoonotic transmission might be predominant in the HIV-positive patient group in the United Kingdom.

Eradication of cryptosporidia and microsporidia following successful antiretroviral therapy.

OBJECTIVES: Incidence of opportunistic protozoal infections causing diarrheal illnesses in patients with HIV has decreased since the introduction of highly active antiretroviral therapy (HAART). The objective of this study was to determine whether the parasites, cryptosporidia, and microsporidia were effectively eradicated or only suppressed following treatment. DESIGN: Six HIV-positive patients with diarrheal symptoms caused by cryptosporidia or microsporidia were prospectively followed up with stool samples and duodenal biopsies. Samples were taken before HAART, between 1 to 3 months, and 6 months post-HAART. METHODS: Duodenal samples were analyzed using routine histology and transmission electron microscopy. Stool samples were analyzed by both light microscopy and polymerase chain reaction (PCR) techniques. RESULTS: Patients who responded successfully to HAART eradicated both cryptosporidial and microsporidial organisms. Symptoms improved within 1 month of therapy but complete eradication of the organisms was only observed after 6 months of treatment. CONCLUSIONS: AIDs-related cryptosporidiosis and microsporidiosis can be cured following successful antiretroviral therapy.

Does Cryptosporidium parvum have a clonal population structure?

Cryptosporidiosis, the disease caused in humans by the opportunistic parasite Cryptosporidium parvum, is the result of zoonotic or anthroponotic transmission. Molecular characterization of different isolates from humans and other mammalian species has recently shown this species to be heterogeneous; this heterogeneity has been linked to the host of isolation, suggesting that the parasites causing zoonotic cryptosporidiosis and those propagated by anthroponotic transmission are genetically distinct. Here, Fatih Awad-El-Kariem provides an update on the taxonomic and epidemiological significance of these observations, and discusses evidence for and against the clonality hypothesis as a model to explain strain variation in this species.

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.

Differentiation between human and animal isolates of Cryptosporidium parvum using molecular and biological markers.

Isolates of Cryptosporidium parvum obtained from infected humans, calves and lambs were typed using arbitrary primed polymerase chain reaction (AP-PCR) and isoenzyme electrophoresis. All animal isolates tested (n = 17) showed similar profiles in AP-PCR and isoenzyme typing. In AP-PCR assays, 9 out of 15 human isolates showed a distinct "human" profile while the remaining 6 isolates showed the "animal" profile. In isoenzyme typing, 5 human isolates which had shown "human" profiles in AP-PCR demonstrated a unique isoenzyme banding pattern, while 2 isolates which had shown "animal" profiles in AP-PCR gave the "animal" banding pattern. In a murine model of infection, all four animal isolates tested were highly infective but only one of four human isolates identified as "human" type in the AP-PCR and isoenzyme typing systems was infective. The good correlation between the data from the different typing systems supports the hypothesis that there are genetically distinct human and animal populations of C. parvum.

Crithidia fasciculata as feeder cells for malaria parasites.

Crithidia fasciculata was used to replace murine peritoneal wash cells as feeder cells for the adaptation of Plasmodium falciparum isolates to continuous culture in vitro, thus avoiding the need to sacrifice animals. Fourteen of 17 malaria parasite isolates in one study, and 12 of 12 isolates in a second study, were successfully adapted to continuous culture in the presence of C. fasciculata, while only 5 of 17 parallel control isolates in the first study, and 2 of 12 isolates in the second study, were adapted in the absence of any feeder cells. Biochemical assays were performed to investigate various hypotheses put forward to explain the mode of action of feeder cells. No effect of C. fasciculata feeder cells was observed on lactate removal, osmotic pressure, or glucose or amino acid content of the malaria culture media. This feeder cell system was shown to reduce the pH of the malaria culture medium. Neither this feeder system nor another system, murine peritoneal macrophages, had any effect on the cysteine content of the culture medium. C. fasciculata was shown to reduce the redox potential of the culture medium, as were other malaria growth enhancers including cysteine and glutathione. This effect on the redox potential of the culture medium is proposed to be a possible mode of action for the feeder cell systems studied.

Differentiation between human and animal strains of Cryptosporidium parvum using isoenzyme typing.

Isoenzyme typing was used to study a number of oocyst isolates of Cryptosporidium parvum from different geographical locations and of human or animal origin. All isolates showed identical enzyme motility when glucose phosphate isomerase (GPI; 23 isolates tested) or lactate dehydrogenases (LDH; 20 isolates tested) was assayed. However, two isoenzyme forms were observed with phosphoglucomutase (PGM; 9 animal isolates showed one form, while 8/9 human isolates showed a second form) and hexokinase (HK; 4 human isolates showed one form and 6 animal isolates showed a second form). Thus, PGM and HK each exhibit 2 isoenzymes corresponding to 2 parasite populations associated with separate hosts. The data from this study, plus supportive evidence obtained by different methods and by independent researchers, lend support to the hypothesis that separate cycles of transmission of C. parvum may exist within human and animal hosts.

Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction.

The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).

Chloroquine-resistant Plasmodium falciparum isolates from the Sudan lack two mutations in the pfmdr1 gene thought to be associated with chloroquine resistance.

Isolates of Plasmodium falciparum from 3 areas of the Sudan were recovered from cryopreservation in London and their chloroquine sensitivity was determined in vitro. Chloroquine resistance was detected in 6/6 isolates from Khartoum, 1/4 from Sennar and 3/3 from Gadarif, indicating that resistance is spreading. All the isolates were sensitive to mefloquine. Studies using blood spots on glass fibre discs and the polymerase chain reaction did not detect two mutations in the pfmdr1 gene, thought to be correlated with chloroquine-resistance, in any of the isolates studied.