Stephanie P. Johnston

Evaluation of Three Commercial Assays for Detection of Giardia and Cryptosporidium Organisms in Fecal Specimens

ABSTRACT There is an increasing demand for diagnostic testing for Giardia intestinalis (G. lamblia) and Cryptosporidium parvum, with a priority being placed on obtaining diagnostic results in an efficient and timely manner. Several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting Giardia and Cryptosporidium. We compared one of these rapid tests, the ImmunoCard STAT! (Meridian Bioscience, Inc.) lateral-flow immunoassay, with the MERIFLUOR direct fluorescent-antibody (DFA) test, the ProSpecT EZ microplate assay for Giardia and the ProSpecT microplate assay for Cryptosporidium, and modified Kinyouns acid-fast stained smears for the detection of Cryptosporidium using 246 specimens. The MERIFLUOR DFA (Meridian Bioscience, Inc.) test detected the largest number of cases (32 Giardia and 37 Cryptosporidium) infections and was used to calculate the sensitivity and specificity of the other tests. For Giardia, the sensitivities of the ImmunoCard STAT! and the ProSpecT Giardia EZ microplate assay (Alexon-Trend, Inc.) were 81 and 91%, respectively. For detection of Cryptosporidium, the sensitivities of the ImmunoCard STAT!, the ProSpecT Cryptosporidium microplate assay (Alexon-Trend, Inc.), and modified Kinyouns acid-fast stained smears were 68, 70, and 78%, respectively. Test specificities were equal to or greater than 99%. Specimens with very small numbers of organisms were not detected by the ImmunoCard STAT!, the ProSpecT microplate assay or modified Kinyouns acid-fast stained smears.

PCR as a Confirmatory Technique for Laboratory Diagnosis of Malaria

ABSTRACT We compared a nested PCR assay and microscopic examination of Giemsa-stained blood films for detection and identification of Plasmodium spp. in blood specimens. PCR was more sensitive than microscopy and capable of identifying malaria parasites at the species level when microscopy was equivocal.

Hospitalization and Mortality Among Primarily Nonbreastfed Children During a Large Outbreak of Diarrhea and Malnutrition in Botswana, 2006

Background:In 2006, a pediatric diarrhea outbreak occurred in Botswana, coinciding with heavy rains. Surveillance recorded a 3 times increase in cases and a 25 fold increase in deaths between January and March. Botswana has high HIV prevalence among pregnant women (33.4% in 2005), and an estimated 35% of all infants under the age of 6 months are not breastfed. Methods:We followed all children <5 years old with diarrhea in the countrys second largest referral hospital at the peak of the outbreak by chart review, interviewed mothers, and conducted laboratory testing for HIV and enteric pathogens. Results:Of 153 hospitalized children with diarrhea, 97% were <2 years old; 88% of these were not breastfeeding. HIV was diagnosed in 18% of children and 64% of mothers. Cryptosporidium and enteropathogenic Escherichia coli were common; many children had multiple pathogens. Severe acute malnutrition (kwashiorkor or marasmus) developed in 38 (25%) patients, and 33 (22%) died. Kwashiorkor increased risk for death (relative risk 2.0; P = 0.05); only one breastfeeding child died. Many children who died had been undersupplied with formula. Conclusions:Most of the severe morbidity and mortality in this outbreak occurred in children who were HIV negative and not breastfed. Feeding and nutritional factors were the most important determinants of severe illness and death. Breastfeeding is critical to infant survival in the developing world, and support for breastfeeding among HIV-negative women, and HIV-positive women who cannot formula feed safely, may prevent further high-mortality outbreaks.

Cryptosporidiosis Associated with Ozonated Apple Cider

We linked an outbreak of cryptosporidiosis to ozonated apple cider by using molecular and epidemiologic methods. Because ozonation was insufficient in preventing this outbreak, its use in rendering apple cider safe for drinking is questioned.

Resistance of Acanthamoeba Cysts to Disinfection in Multiple Contact Lens Solutions

ABSTRACT Acanthamoebae are free-living amoebae found in the environment, including soil, freshwater, brackish water, seawater, hot tubs, and Jacuzzis. Acanthamoeba species can cause keratitis, a painful vision-threatening infection of the cornea, and fatal granulomatous encephalitis in humans. More than 20 species of Acanthamoeba belonging to morphological groups I, II, and III distributed in 15 genotypes have been described. Among these, Acanthamoeba castellanii, A. polyphaga, and A. hatchetti are frequently identified as causing Acanthamoeba keratitis (AK). Improper contact lens care and contact with nonsterile water while wearing contact lenses are known risk factors for AK. During a recent multistate outbreak, AK was found to be associated with the use of Advanced Medical Optics Complete MoisturePlus multipurpose contact lens solution, which was hypothesized to have had insufficient anti-Acanthamoeba activity. As part of the investigation of that outbreak, we compared the efficacies of 11 different contact lens solutions against cysts of A. castellanii, A. polyphaga, and A. hatchetti (the isolates of all species were genotype T4), which were isolated in 2007 from specimens obtained during the outbreak investigation. The data, generated with A. castellanii, A. polyphaga, and A. hatchetti cysts, suggest that the two contact lens solutions containing hydrogen peroxide were the only solutions that showed any disinfection ability, with 0% and 66% growth, respectively, being detected with A. castellanii and 0% and 33% growth, respectively, being detected with A. polyphaga. There was no statistically significant difference in disinfection efficacy between the 11 solutions for A. hatchetti.

Case-control study to determine risk factors for diarrhea among children during a large outbreak in a country with a high prevalence of HIV infection.

OBJECTIVES Between January and March of 2006, over 35 000 diarrhea cases and 532 deaths were reported among children aged <5 years in Botswana. We conducted an investigation to characterize the outbreak, identify risk factors for diarrhea, and recommend control strategies. METHODS We enrolled children <5 years of age presenting to the emergency department between March 2 and March 20, 2006. Cases had ≥3 loose stools per day and no antecedent diarrhea among household members. Controls had had no diarrhea since January 1, 2006. We conducted a multivariate logistic regression analysis controlling for socioeconomic status, age, and maternal HIV status. RESULTS Forty-nine cases with median age of 12 months (range 0-45 months) and 61 controls with median age of 24 months (range 0-59 months) were enrolled; 33 (30%) were born to HIV-positive mothers. Case-parents were more likely to report storing household drinking water (adjusted odds ratios (AOR) 3.9, 95% confidence interval (CI) 1.2-15.7). Lack of hand washing after using the toilet or latrine (AOR 4.2, 95% CI 1.1-20.4) was more likely to be reported by case-parents. Case-children were less likely to be currently breastfeeding (AOR 30.3, 95% CI 2.0-1000.0). Five (10%) case-patients and no control-patients died. Multiple causal pathogens were identified. CONCLUSIONS During this diarrhea outbreak in a country with a national program to prevent mother-to-child transmission of HIV, ill children were less likely to be breastfed and more likely to have been exposed to environmental factors associated with fecal contamination. These findings underscore the importance of adequate access to safe water, sanitation, hygiene, and nutrition education among populations using breast milk substitutes.

Rapid Microsphere Assay for Identification of Cryptosporidium hominis and Cryptosporidium parvum in Stool and Environmental Samples

ABSTRACT Cryptosporidium hominis and Cryptosporidium parvum are associated with massive disease outbreaks worldwide. Because these two species have different transmission cycles, identification of these parasites to the species level in clinical samples may provide laboratory data of crucial importance in epidemiologic investigations. To date, the most reliable way to differentiate C. hominis and C. parvum is based on DNA sequencing analysis of PCR amplicons. Although this approach is very effective for differentiation of Cryptosporidium species, it is labor-intensive and time-consuming compared with methods that do not require DNA sequencing analysis as an additional step and that have been successfully used for specific identification of a number of pathogens. In this study, we describe a novel Luminex-based assay that can differentiate C. hominis from C. parvum in a rapid and cost-effective manner. The assay was validated by testing a total of 143 DNA samples extracted from clinical specimens, environmental samples, or samples artificially spiked with Cryptosporidium oocysts. As few as 10 oocysts per 300 μl of stools could be detected with this assay. The assay format includes species-specific probes linked to carboxylated Luminex microspheres that hybridize to a Cryptosporidium microsatellite-2 region (ML-2) where C. hominis and C. parvum differ by one nucleotide substitution. The assay proved to be 100% specific when samples that had been characterized by direct fluorescent antibody test (DFA) and DNA sequencing analysis were tested. In addition, the assay was more sensitive than DFA and provided species identification, which is an advantage for epidemiologic studies.

Subtype Analysis of Cryptosporidium Specimens from Sporadic Cases in Colorado, Idaho, New Mexico, and Iowa in 2007: Widespread Occurrence of One Cryptosporidium hominis Subtype and Case History of an Infection with the Cryptosporidium Horse Genotype

ABSTRACT Subtyping was conducted in late 2007 on 57 Cryptosporidium specimens from sporadic cases in Colorado, Idaho, New Mexico, and Iowa. One previously rare Cryptosporidium hominis subtype was indentified in 40 cases (70%) from all four states, and the Cryptosporidium horse genotype was identified in a pet shop employee with severe clinical symptoms.

Multiple risk factors associated with a large statewide increase in cryptosporidiosis

Cryptosporidium species have emerged as a major cause of outbreaks of diarrhoea and have been associated with consumption of contaminated recreational and drinking water and food as well as contact with infected attendees of child-care programmes. In August 2007, the Colorado Department of Public Health and Environment detected an increase in cryptosporidiosis cases over baseline values. We conducted a case-control study to assess risk factors for infection and collected stool specimens from ill persons for microscopy and molecular analysis. Laboratory-confirmed cases (n=47) were more likely to have swallowed untreated water from a lake, river, or stream [adjusted matched odds ratio (aOR) 8.0, 95% confidence interval (CI) 1.3-48.1], have had exposure to recreational water (aOR 4.6, 95% CI 1.4-14.6), or have had contact with a child in a child-care programme or in diapers (aOR 3.8, 95% CI 1.5-9.6). Although exposure to recreational water is commonly implicated in summertime cryptosporidiosis outbreaks, this study demonstrates that investigations of increased incidence of cases in summer should also examine other potential risk factors. This study emphasizes the need for public health education efforts that address the multiple transmission routes for Cryptosporidium and appropriate prevention measures to avoid future transmission.

Giardiasis outbreak at a camp after installation of a slow-sand filtration water-treatment system.

In July and August 2007, a giardiasis outbreak affected attendees of a private recreational camp in California. Twenty-six persons had laboratory-confirmed giardiasis; another 24 had giardiasis-like illness with no stool test. A retrospective cohort study determined that showering was associated with illness (adjusted odds ratio 3·1, 95% confidence interval 1·1-9·3). Two days before the outbreak began, the camp had installed a slow-sand water filtration system that included unsterilized sand. Review of historical water-quality data identified substantially elevated total coliform and turbidity levels in sand-filtered spring water used for showering during the suspected exposure period. Unfiltered spring water tested at the same time had acceptable coliform and turbidity levels, implicating the filtration system as the most likely contamination source. To prevent waterborne illness, slow-sand water filtration systems should use sterilized sand, and slow-sand-filtered water should not be used for any purpose where inadvertent ingestion could occur until testing confirms its potability.