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An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Giardia lamblia in human feces. The assay could detect between 37 and 375 trophozoites from cultures and between 12.5 and 125 cysts purified from human stool. Stool specimens were positive by ELISA in 36 (92%) of 39 patients with giardiasis; negative specimens came from patients with low numbers of parasites in their stool. In 10 patients stools became negative by ELISA after successful treatment. Stool specimens were positive by ELISA in three (2%) of 128 patients without demonstrable Giardia organisms in their stools; one positive specimen was from a child with IgA deficiency and diarrhea who responded clinically to metronidazole. The ELISA is a simple, sensitive, and specific diagnostic test for G lamblia that will be useful in diagnosis, in follow-up treatment, and in large-scale studies directed at defining the epidemiology and pathophysiology of G lamblia infection. [ABSTRACT FROM AUTHOR]

In animal and clinical studies of Cryptosporidium infection, counting Cryptosporidium oocysts in collected fecal samples is often critical to interpretation of results and validity of statistical analyses. In this experiment, 3 methods of counting oocysts derived from feces of infected mice were compared. These included sequential counting of oocysts in 25 adjacent microscopic fields, random counting of oocysts in 25 scattered microscopic fields, and hemacytometer counting of oocysts using standard procedures. Numerical results are expressed as the numbers of oocysts per microscopic field for sequential and random counting methods or per microliter for the hemacytometer method. Our results showed that the numbers of oocysts adjusted for volume and weight, or for volume alone, were similar for all 3 techniques. Linear relationships between the 3 methods allow use of linear regression to convert the number of oocysts counted by 1 method into those counted by another. This suggests that comparisons between different experiments in which Cryptosporidium oocysts are counted using any of these methods are both possible and valid.

Geographic variation in the HIV-1 virus is extensive but incompletely documented. We herein report the first genetic characterization of HIV-1 isolates from Zambia. The genomic region encoding the GAG polyprotein has been compared among 22 Zambian isolates and 14 North American isolates using a combination of polymerase chain reaction (PCR) and DNA sequencing methods. The Zambian isolates were similar to one another but distinct from other HIV-1 isolates. They exhibited a characteristic PCR "fingerprint" wherein certain primer combinations were unable to amplify because of mispairing. The sequence of the complete gag gene of three isolates from Zambia has been determined, and phylogenetic tree analysis placed them in a branch distinct from other African isolates and North American isolates. The PCR procedure used here may be widely applicable for genetic characterization of HIV-1.

An unidentified organism was found in the stools of 55 immunocompetent patients who presented to the CIWEC Clinic in Kathmandu, Nepal between June and November 1989. The microscopic features of the organism share characteristics of both coccidia and cyanobacteria species. From June 26, 1989 to November 17, 1989, 55 persons were identified as having the organism in at least one stool sample. The illness was characterized by prolonged watery diarrhea, anorexia, fatigue, and weight loss. The mean +/- SD duration of illness was 43 +/- 24 days (range 4-107). Thirty-four patients received a total of 78 courses of antimicrobial treatment (2.3 courses/patient). The mean +/- SD duration of illness in 34 treated patients was 46 +/- 24 days. In 14 untreated patients, the mean +/- SD duration of illness was 35 +/- 23 days. The organism is 8.0-9.0 microns in diameter, floats in Sheather's solution, and stains red with the modified acid-fast stain. Since the agent was closely associated with a prolonged, self-limited diarrheal illness, it could easily have been misdiagnosed as Cryptosporidium. The organism should be looked for in the stools of patients with persistent diarrhea and a history of foreign travel.

Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3'LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3' end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3' LTR segments of the genome were used to compare sequential HIV-1 isolates form six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1 isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.

Cryptosporidium is a parasite of the human gastrointestinal tract that can cause life-threatening diarrhea in immunodeficient patients. Although more than 80 agents have been tried with occasional anecdotal success, treatment remains primarily limited to hydration. A 38-yr-old homosexual man with antibody to human immunodeficiency virus and Cryptosporidium-related diarrhea is described. The patient excreted 6-12 L of stool per day for at least 3 mo, 2 of them spent in the hospital. Trials with more than 6 antidiarrheal medications were ineffective. The patient received bovine colostrum hyperimmune to Cryptosporidium by direct duodenal infusion. During infusion, the patient's fecal output decreased to less than 2 L per day, and 48 h after treatment, stools were formed and oocysts to Cryptosporidium were absent. The patient remained asymptomatic for 3 mo. Hyperimmune bovine colostrum offers an exciting new therapy for cryptosporidiosis; controlled trials to establish efficacy should be undertaken and the active factor(s) characterized.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy-and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool.

A bacterial pathogen was isolated from 47% of 328 expatriate patients with diarrhea seen at two medical clinics in Nepal in 1986. Enterotoxigenic Escherichia coli (24%), Shigella (14%), and Campylobacter species (9%) were isolated most frequently. Enteroinvasive and adherence factor-positive E coli were isolated from 2% and 1% of patients, respectively. Giardia lamblia was detected in 12% of patients, rotavirus in 8%, and Cryptosporidium and Entamoeba histolytica each in 5%. Blastocystis hominis was present in 33% of patients but in only 9% of those who took trimethoprim-sulfamethoxazole. More than one enteropathogen was detected in 17% of patients. Patients with prolonged symptoms (longer than two weeks) were more likely to have Giardia (27%) and less likely to have Shigella (5%) than were patients with acute symptoms. The isolation rates of bacterial pathogens decreased with length of stay in Nepal. A wide variety of enteropathogens were detected in travelers to Nepal, and Shigella and protozoa were particularly important. Length of time abroad and duration of symptoms were important diagnostic considerations.

Between January 12 and February 7, 1987, an outbreak of gastroenteritis affected an estimated 13,000 people in a county of 64,900 residents in western Georgia. Cryptosporidium oocysts were identified in the stools of 58 of 147 patients with gastroenteritis (39 percent) tested during the outbreak. Studies for bacterial, viral, and other parasitic pathogens failed to implicate any other agent. In a random telephone survey, 299 of 489 household members exposed to the public water supply (61 percent) reported gastrointestinal illness, as compared with 64 of 322 (20 percent) who were not exposed (relative risk, 3.1; 95 percent confidence interval, 2.4 to 3.9). The prevalence of IgG to cryptosporidium was significantly higher among exposed respondents to the survey who had become ill than among nonresident controls. Cryptosporidium oocysts were identified in samples of treated public water with use of a monoclonal-antibody test. Although the sand-filtered and chlorinated water system met all regulatory-agency quality standards, sub-optimal flocculation and filtration probably allowed the parasite to pass into the drinking-water supply. Low-level cryptosporidium infection in cattle in the watershed and a sewage overflow were considered as possible contributors to the contamination of the surface-water supply. We conclude that current standards for the treatment of public water supplies may not prevent the contamination of drinking water by cryptosporidium, with consequent outbreaks of cryptosporidiosis.

Rabbit antisera to 11 Giardia lamblia isolates were reacted with 8 G. lamblia isolates using single antibody ELISA and indirect immunofluorescent antibody techniques (IFA). Using living trophozoite organisms, IFA showed marked surface fluorescence with homologous antisera-organism pairs while heterologous pairs showed reduced or no reactivity. Using formalin-fixed trophozoites, the pattern of fluorescence changed to include diffuse internal fluorescence with both homologous and heterologous pairs. In contrast to the variability in surface fluorescence, similar reactivity was noted for homologous and heterologous antisera-isolate pairs with the ELISA. In addition, a double antibody enzyme immunoassay using rabbit antisera prepared to 2 G. lamblia isolates was performed with 8 different isolates as test antigen. All 8 were equally well detected. These data confirm previous findings that G. lamblia isolates have both different and common antigens. The different antigens appear to be on the surface of the organism while the common antigens appear to be internal or somatic.