Readers should systematically assess methods used to identify, measure and analyze confounding in observational cohort studies.

OBJECTIVE: To describe techniques used to address confounding in published observational studies. STUDY DESIGN AND SETTING: A systematic literature review identified studies using administrative or registry data to investigate health effects of drug therapies. Studies published from January 2001 to December 2005 came from BMJ, New England Journal of Medicine, Lancet, Annals of Internal Medicine, and JAMA. A structured abstraction form was used to collect information about confounding. RESULTS: The search identified 29 studies. Twenty-two studies (76%) had 10,000 or more subjects and 18 (62%) used a mortality outcome. None mentioned use of a literature search to identify confounders, however, 28 (97%) listed confounders included, and 26 (90%) listed confounders not included in the study. Eighteen (62.1%) discussed the validity of confounder data. Most (22, or 76%) studies included a table with the distribution of confounders but none used effect size to assess imbalance between comparison groups. Almost all studies used regression techniques (28, or 97%); fewer used stratification (16, or 55%) or matching (four, or 14%) to address confounding. Eleven (40%) studies discussed sensitivity analyses. CONCLUSION: Published cohort studies routinely include a list of potential confounders but there is room for improvement in confounder identification, measurement, and analysis.

Age, sex, and household exposure are associated with the acute measles-specific antibody-dependent cellular cytotoxicity antibody response.

To determine whether functional antibody responses correlate with factors associated with severe measles, measles-specific antibody-dependent cellular cytotoxicity (ADCC) and neutralizing antibodies were measured in 114 Filipino children with measles. Children > 24 months old were more likely to have ADCC antibody in acute sera than were those < or = 24 months (odds ratio = 3.6, 95% confidence interval = 1.7-7.8). This age-related difference in ADCC prevalence was most apparent between younger and older girls. Among children < or = 24 months, a higher prevalence of ADCC antibody was associated with male sex, absence of lymphopenia, and household exposure to measles. The presence of ADCC antibody was not associated with malnutrition or diarrhea. Neutralizing antibody titers were lower in children with lymphopenia but showed no relationship with the other variables. Thus, the ADCC antibody response is associated with some risk factors related to measles severity. Attenuation of this response may contribute to the severity of infection.

Statistical analysis of clinical, immunological and nutritional factors in pediatric cryptosporidiosis in the Philippines.

A statistical analysis of clinical, nutritional, and immunological data gathered in a previous study suggest that nutritional factors, and in particular, iron status, appeared to be of significance in mounting an effective immune response to Cryptosporidium infection in young children. The primary protective mechanism seemed to be cell-mediated; humoral immunity was intact in all the study subjects, however, CMI was initially impaired but improved over six weeks.

Migration of 75Se-methionine-labeled Schistosoma japonicum in normal and immunized mice.

The patterns of migration and attrition of Schistosoma japonicum larvae were studied in a mouse model. Control and immunized mice were challenged with 100 S. japonicum cercariae tagged with 75Se-labeled methionine. Skin, lungs, liver, and other organs were analyzed by compressed organ autoradiography for the presence of larvae that appeared as reduced silver foci. The pattern of migration of S. japonicum was similar in mice with primary infection and in mice immunized with irradiated cercariae. Skin was not a site of attrition after primary infection nor after immunization. Attrition occurred after migration to the lungs and continued until after migration to the liver in mice with primary infection, while in immunized mice attrition occurred before lung migration and continued at a faster rate than in normal mice. In both control and immunized mice, the lungs and liver were the major sites of attrition.

Detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue by the polymerase chain reaction.

The objective of this project was to demonstrate detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue using the polymerase chain reaction (PCR). DNA was purified from six samples of fixed, paraffin-embedded tissue that were histologically positive for C. parvum and used in the PCR. Previously developed oligonucleotide primers specific for C. parvum were used to amplify a 452-base target sequence, and a 20-base synthetic probe labeled with digoxigenin-11-dUTP was used to detect the amplification product by chemiluminescence. All six samples were positive by PCR; negative controls showed no amplification or hybridization. This approach could provide a sensitive and specific method for detection of parasite material in fixed, paraffin-embedded tissue samples, and prove to be of significant value in retrospective studies of archival material.

DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction.

The objective of this project was to construct specific and sensitive molecular probes and amplification primers for Cryptosporidium parvum that could be used in diagnosis, retrospective tissue studies, and in epidemiologic surveys. Whole genomic DNA was extracted from oocysts of C. parvum purified from human and bovine feces. A genomic library was constructed in plasmid pUC18 and propagated in Escherichia coli DH5 alpha. Transformants were screened by colony hybridization and autoradiography. The 2.3-kilobase segment in plasmid pHC1, a clone specific for C. parvum, was sequenced by the Sanger method. Computer analysis gave a G+C content of 35%. A 400-base region (bases 470-870) was selected as an amplification target because it contained a unique restriction endonuclease site that could serve as a useful marker. Primers of 26 nucleotides each were synthesized. Sensitive and specific amplification of the target sequence was demonstrated both by ethidium bromide staining of agarose and acrylamide gels, and by hybridization with chemiluminescence-labeled synthetic oligonucleotide probes.

Immune response to cryptosporidiosis in Philippine children.

An ELISA was used to measure the Cryptosporidium-specific IgA, IgG, and IgM antibody levels in serum, stool, and duodenal fluid of 15 Filipino children. Antibody levels were measured on admission to the hospital, 1 week later, and at a 6 week follow-up examination. Delayed type hypersensitivity skin tests were used to assay cell mediated immunity (CMI), iron status was measured by serum iron tests and total iron binding capacity, and the degree of malnutrition was determined by clinical examination. Antibody response to Cryptosporidium was qualitatively and quantitatively strong and maintained over time. All subjects showed impaired CMI early with some reconstitution after 6 weeks. All subjects showed some degree of malnutrition and/or depleted iron status.

Production of monoclonal antibodies specific for Eimeria tenella microgametocytes.

A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.

Intramolecular hydrogen-bonding in beta adrenergic blocking agents.

The acid dissociation constants for nine beta-adrenergic blocking agents have been determined. Their unusually low pK(a), values are explained as being due to the formation of an intramolecular hydrogen bond, between the terminal amino group and the beta-hydroxyl group on the alkanolamine side-chain which is common to this class of drugs.