Simultaneous administration of multiple model substrates to assess hepatic drug clearance.

We have evaluated a method to simultaneously assess three major processes involved in hepatic drug clearance using three model substrates administered simultaneously as a 5-minute intravenous injection. Lorazepam, indocyanine green, and antipyrine are used to assess conjugation, liver blood flow, and microsomal oxidative metabolism, respectively. These substrates were administered individually and as a mixture to 10 healthy adult male volunteers to determine if clearances of any of the compounds were affected by simultaneous administration. Mean clearances of the substrates were not different when administered alone (9.97, 0.78, and 0.53 ml/min/kg) vs. together (11.5, 0.89, and 0.52 ml/min/kg), using a paired t test. Since we were using this method to assess hepatic drug clearance in children with leukemia, the effect of short-term allopurinol was assessed. The three model substrates were administered to the volunteers after 0, 1, 8, and 22 days of treatment with allopurinol, 200 mg t.i.d. There was no change in mean clearance of any of the three compounds at any point during allopurinol treatment (repeated-measures ANOVA). We conclude that this technique is a simple and valid method to simultaneously assess three major processes involved in hepatic drug clearance and is not affected by up to 22 days of oral allopurinol treatment. This simple technique, requiring a single set of blood samples, has potential applications in the assessment of developmental changes in hepatic drug clearance, as well as the effects of environmental, therapeutic, and pathophysiologic factors on three major processes involved in hepatic drug clearance.

Determinants of trachoma endemicity using Chlamydia trachomatis ompA DNA sequencing.

A six-year prospective study of Chlamydia trachomatis infection and ocular disease in Tanzanian village children was conducted to identify the determinants of trachoma endemicity using sequencing of ompA. Overall, 749 conjunctival samples were obtained, with 176 children sampled in both 1989 and 1995. 31.1% (233/749) were positive by PCR-enzyme immunoassay, and 76% (176/233) of the positives were sequenced in variable domains (VD) 1 to 4 (22 children in both 1989 and 1995). Twenty-six ompA genotypes of serovar A, and 19 of B/Ba were identified, and only 20% of genotypes identified in 1995 matched those found in 1989. In particular, B/Ba genotypes exhibited a 15-base region in VD 2 with increased nucleotide substitution, and these types were associated with age and water availability. Homotypic infection and infection with multiple genotypes and high chlamydial load did predict subsequent severe trachoma (odds ratio (OR) = 10.14, 95% confidence interval (CI): 1.71, 60.23; OR = 6.40, 95% CI: 0.75, 54.41; OR = 6.74, 95% CI: 0.82, 55.38, respectively). And, multitypic infection was clustered with residence of village and associated with familial cattle ownership. In conclusion, high ompA polymorphism and the inability of some hosts to clear infection with the same ompA genotype suggest two distinct but converging mechanisms of endemic severe trachoma.

Nonocular Chlamydia infection and risk of ocular reinfection after mass treatment in a trachoma hyperendemic area.

PURPOSE: The presence of nasal discharge on a child's face increases the risk of active trachoma, suggesting that Chlamydia trachomatis in nasal secretions may be a possible source of ocular reinfection. The prevalence of chlamydia in nasal secretions and the risk of reinfection after mass treatment was investigated in a hyperendemic area of Tanzania. METHODS: In one village a total of 232 children aged 1 to 7 years were followed before and after mass treatment. Clinical trachoma, and microbiologic evidence of chlamydia, were assessed at baseline, 2 and 4 weeks into mass treatment, and 4 weeks after treatment stopped. The presence of chlamydia in ocular and nasal secretions was determined by polymerase chain reaction-enzyme immunoassay techniques. RESULTS: Of the 232 children, 59% had clinical trachoma and 27% had nasal specimens positive for chlamydia. Children with positive ocular chlamydia specimens and/or clinical trachoma were significantly more likely to have positive nasal specimens. At the end of mass treatment, only 4% of children had positive ocular specimens. However, 1 month after treatment stopped, the incidence of new infection was 21%. The rate of new ocular infections in those who had negative ocular specimens after treatment was similar between those who had positive and those who had negative nasal specimens at baseline. Positive ocular specimens at baseline was not a predictor of risk of new infection after treatment (odds ratio = 1.18, 95% confidence interval = 0.58, 2.40), suggesting these new infections were not the result of latent or persistent organism. CONCLUSIONS: These data do not support a role for nasal secretions in causing reinfection after treatment. One mass topical treatment alone is unlikely to be effective in trachoma hyperendemic areas as shown by the rapid re-emergence of infection.

Enzyme immunoassay for detection of hybrids between PCR-amplified HIV-1 DNA and a RNA probe: PCR-EIA.

An enzyme immunoassay was developed to detect human immunodeficiency virus type 1 (HIV-1) DNA amplified by polymerase chain reaction (PCR-EIA). A set of primers (outer set) was used in PCR to amplify a segment of the HIV-1 gag gene from peripheral blood mononuclear cells. Hybrids between the amplified DNA and a RNA probe were measured in a microtiter plate immunoassay using a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids and a fluorescent substrate. A second set of primers (nested set) located within the outer set was used in PCR with a known template to prepare the probe. One primer of the nested set included the T7 RNA polymerase promoter at its 5' end allowing transcription of a single-stranded RNA probe. Ten copies of HIV-1 DNA could be detected by PCR-EIA (42 fluorescent units with a background of 18 fluorescent units) compared with a detection limit of 1000 copies by ethidium bromide-stained agarose gel. HIV-1 DNA was detected by PCR-EIA in peripheral blood mononuclear cells from 32 of 33 seropositive patients (range 54-810 fluorescent units), and 0 of 25 seronegative patients (range 20-40 fluorescent units) (sensitivity 97%; specificity 100%). PCR-EIA offers a practical and nonisotopic method to objectively measure PCR-amplified HIV-1 DNA and has the potential for the measurement of other microbial pathogens in human body fluids.

Incidence of trichiasis in a cohort of women with and without scarring.

BACKGROUND: Blindness from trachoma is a significant problem for many underdeveloped countries. While active trachoma is common in children, trichiasis, the potentially blinding sequella, develops in adulthood and affects mainly women. Little is known about factors associated with the development of trichiasis. METHODS: The 7-year incidence of trichiasis and its association with ocular chlamydia infection was examined in a cohort of women from a hyperendemic area. A total of 4,932 women 18 years and older, living in 11 villages in Central Tanzania, were examined in 1989. A follow-up examination in 1996 was performed on all women with scars living in six of the 11 villages and on a random sample of women without scars from the same villages. Trachoma was graded clinically, chlamydia infection was ascertained at follow-up using polymerase chain reaction-enzyme immunoassay (PCR-EIA). RESULTS: A total 523 of the women with scars and 503 of the women without scars were re-examined. Forty-eight of the women with scars (incidence, 9.2%) and three of the women without scars (0.6%) developed trichiasis in the 7-year period. Prevalence of chlamydia infection was significantly higher in the group with scars (11.7% versus 7.1%). Trichiasis cases were more likely to be older, and to have chlamydia infection at follow-up odds ratio (95% confidence interval) 2.5 (1.1-5.7). CONCLUSION: The 7-year incidence rate in the population with scars was high, over 1% per year. Ocular chlamydia infection was more common in the group with scars at baseline and was also associated with being a trichiasis case, suggesting the importance of potentially long-term chlamydia infection in the progression to trichiasis. Antibiotic distribution programmes for trachoma control should include women with scars.

Effects of a protective foam on scrubbing and gloving.

OBJECTIVE: To test the effects of a skin protectant on surgical scrub and glove integrity. DESIGN: Forty-nine healthy adult volunteers were assigned (12 subjects per group) to apply a protective foam (DermaMed; Benchmark Enterprises, Salt Lake City, Utah) in conjunction with surgical scrub in one of the following formulations: 70% isopropyl alcohol, a liquid detergent base containing 4% chlorhexidine gluconate, a liquid detergent base containing 7.5% povidone-iodine, or a nonantimicrobial liquid soap (control). According to a standard protocol, subjects performed a surgical scrub on 3 days (every other day). Foam was applied after surgical scrub on day 1 and before surgical scrub on day 3. No foam was applied on day 2. Subjects were gloved for 2 hours after surgical scrub. SETTING: Laboratory setting. RESULTS: On all test days, there were significant differences in bacterial reduction by products (chlorhexidine gluconate or alcohol > povidone-iodine > control). When controlling for baseline counts and products used, there were no significant differences in colony-forming unit counts on hands with or without foam immediately after scrubbing or at 2 hours after scrub on gloved or ungloved hands, nor were there differences in glove leakage rates when foam was on hands. CONCLUSIONS: Such protectants can be used without detrimental effects to scrub effectiveness or glove integrity.

Lack of care giver hand contamination with endemic bacterial pathogens in a nursing home.

Prevalences of Clostridium difficile and multiply resistant Staphylococcus aureus (MRSA) were determined in nursing staff and residents of a 233-bed long-term care facility. Twenty of 38 (52.6%) patients in the long-term care ward and three of 69 (4.3%) in the skilled-nursing ward were colonized with MRSA; 16 of 48 (33%) patients in the long-term care ward and seven of 52 (13%) in the nursing home ward were colonized with C. difficile. None of the 79 staff members whose hands were cultured had chronic C. difficile hand carriage and MRSA was present on only three of 79 (3.9%). Over a 6-month period, 128,000 pairs of gloves were worn. Since C. difficile and MRSA are rarely present on washed hands of care providers, appropriate handwashing and gloving should make a significant contribution to reducing the spread of these agents in long-term care facilities.

Detection of Chlamydia trachomatis DNA in archival paraffinized specimens from chronic salpingitis cases using the polymerase chain reaction.

OBJECTIVE: To identify Chlamydia trachomatis by the polymerase chain reaction (PCR) in fallopian tube tissues with chronic salpingitis. DESIGN: Retrospective case-control study. SETTING: Academic tertiary institution. PATIENT(S): Women with a pathological diagnosis of chronic salpingitis or normal fallopian tube hospitalized between September 1992 and November 1994. Initial identification of 248 specimens with final analysis of 154. INTERVENTION(S): Paraffin-embedded fallopian tube tissues were analyzed with use of PCR to detect C. trachomatis. MAIN OUTCOME MEASURE(S): Identification of C. trachomatis DNA; demographics of age, ethnicity, parity, history of sexually transmitted disease, and surgical procedure. RESULT(S): C. trachomatis DNA was detected in 9 of 77 chronic salpingitis cases. Seventy-seven controls were negative for C. trachomatis. No statistically significant difference in age or ethnicity between cases and controls was identified. Nulliparity was more frequent in cases (26 of 74) than controls (14 of 76). Sexually transmitted disease history was more prevalent in cases (24 of 74) than controls (6 of 76). Chlamydia infection was not associated with a particular surgical indication. CONCLUSION(S): Chronic salpingitis is highly associated with the presence of C. trachomatis infection as detected by PCR.

OmpA genotypic evidence for persistent ocular Chlamydia trachomatis infection in Tanzanian village women.

BACKGROUND: Trachoma is still a significant problem in the developing world. Adult women are at higher risk of developing scarring and trichiasis, the potentially blinding sequelae, compared to men. In part, the higher risk may be due to more frequent infections in women because of their frequent contact with children, the main reservoir of C. trachomatis infection. However, other factors associated with infection, particularly constant infection, in adult women need to be identified. METHODS: A group of 118 women who were infected with C. trachomatis and 118 women who were not infected, but of similar age and trachoma status, were identified in 1996 from a population-based sample of women age 16 and older from eleven villages in Kongwa, Tanzania. This group of 236 was re-contacted three years later to ascertain trachoma status and determine infection status using polymerase chain reaction-enzyme immunoassay (PCR-EIA). Positive samples at both time points were examined for serovar and genotype shift, using ompA sequencing information. RESULTS: Of the original 236 women, 165 (70%) completed exams in 1999. Fifty-eight (35%) of the 165 women were excluded from this analysis because they received antibiotic treatment for trachoma in the six months prior to the second exam. Infection at baseline was the most important predictor of infection three years later (Age-adjusted odds ratio (95% confidence interval) 6.6 (1.8-24.4)). A total of 17 women (16%) were infected at the two examinations, and of the 15 for whom genotyping could be done, 11 (73%) were infected with the same ompA genotype at both time points. Chronically infected women were more likely to have trichiasis, scarring, and active trachoma at baseline than those never infected or infected only once. Only 41% of the chronically infected women were living in houses with infected pre-school children, but 24% were in houses with no children. Four of ten women with trichiasis developed incident corneal opacity over the three years. CONCLUSIONS: The data provide evidence for persistence of infection in a sub-group of women. The strongest predictor of infection at follow-up was baseline infection, and most were infected with the same genotype at both time points. For women with persistent infection, at least half were either not living with children or not living with infected children, suggesting that continual re-exposure from a close family member was less likely. Chronic infection is likely related to both exposure and immunological factors, and these need to be further identified. Inclusion of women in community-based treatment programs, regardless of whether a child is present in the house, is likely to be important in preventing the progression of inflammatory trachoma and scarring to trichiasis.

Principles of intercultural medicine in an internal medicine program.

Internal medicine and medicine-pediatric residents (n = 76) completed a questionnaire that measured variables including sociodemographics, family dynamics, cross-cultural exposure, and exposure to intercultural medicine principles. Questions were answered regarding perceptions of their patients and level of comfort discussing specific cultural variables. Gender, training status, and geographic background did not influence responses, but the responses of European-Americans (71%) vs. ethnic minorities and foreign medical graduates (29%) were significantly different. European-Americans were more likely to be men, less likely to have an urban background (p = .02), and their self-described socioeconomic status was uppermiddle to upper class (p = .02). European-Americans vs. all others differed in their perceptions of patients' financial support (p = .001), and reasons for doctor-patient miscommunications (p = .05). The European-Americans had significantly less exposure to friends and classmates (p = .002), and instructors (p = .0001) of ethnic origins different than their own prior to residency training. Our data support the inclusion of intercultural medicine principles in the general internal medicine curriculum.

Cultural issues in serving minority populations: emphasis on Mexican Americans and African Americans.

Patients' cultural beliefs may affect acceptance of health care, compliance, and treatment outcomes. This article discusses cultural views of health and illness, folk beliefs and customs, cultural barriers to care, and alternative health-care systems, with particular emphasis on Mexican Americans and African Americans, including curanderismo, rootwork, and voodoo. Physicians who wish to provide appropriate and acceptable care in a cross-cultural setting should integrate these beliefs with conventional medicine.

Effective hand degerming in the presence of blood.

This study evaluated the effect of blood on the antimicrobial activity of several agents used for handwashing. Seventy-one healthy adult volunteers used 1 of 6 products (70% isopropyl alcohol [IPA]; liquid hand rinse containing 70% ethyl alcohol and 0.5% chlorhexidine gluconate [EA]; detergent containing 7.5% povidone-iodine [PI]; detergent containing 4% chlorhexidine gluconate [CHG]; a nonantimicrobial soap; and a control group that used no product) in two tests: with and without 1.2 mL of dried sterile sheeps' blood on the hands. In the presence of blood, the two alcohol products (IPA and EA) resulted in significantly greater reductions in numbers of colony-forming units than other products (P less than 0.001). When no blood was present, IPA was associated with significantly greater reductions, whereas soap and control groups had significantly lower reductions (P = 0.008). We conclude that hands are effectively degermed with a variety of products in the presence of blood, and that alcohols give greater initial reductions in colonizing flora. This is of particular relevance in emergency situations during which contamination with blood is likely and handwashing facilities are inaccessible.

Prismatic Metropolis: Race and Residential Segregation in the City of the Angels

Most major urban areas remain segregated by race, especially in terms of black segregation from whites. We replicate and extend the innovative approach developed by Farley and colleagues for understanding processes of racial residential segregation with data collected in Los Angeles. Using a large (N = 4025) multiracial sample of adults, we examine (1) actual and perceived differences in economic status, (2) mutual preference for same race neighbors, and (3) racial prejudice and discrimination as hypotheses for the persistence of residential segregation. With a systematic experimental design we gauge respondent openness to living in areas with varying proportions of black, white, Latino, or Asian neighbors. We find no support for actual or perceived cost of housing as a barrier to integration. Although all groups exhibit some degree of ethnocentric preference for same race neighbors, this tendency is strongest among whites rather than blacks and plays only a small role in perpetuating segregation. Blacks face the greatest hostility in the search for housing and are consensually recognized as most likely to face discrimination in the housing market. Racial minorities are more open to sharing residential space with whites than with other minorities. We find generally higher rates of openness to integration than Farley and colleagues found in their recent Detroit survey.

The ecology and pathobiology of Clostridium difficile infections: an interdisciplinary challenge.

Clostridium difficile is a well recognized pathogen of humans and animals. Although C. difficile was first identified over 70 years ago, much remains unknown in regards to the primary source of human acquisition and its pathobiology. These deficits in our knowledge have been intensified by dramatic increases in both the frequency and severity of disease in humans over the last decade. The changes in C. difficile epidemiology might be due to the emergence of a hypervirulent stain of C. difficile, ageing of the population, altered risk of developing infection with newer medications, and/or increased exposure to C. difficile outside of hospitals. In recent years, there have been numerous reports documenting C. difficile contamination of various foods, and reports of similarities between strains that infect animals and strains that infect humans as well. The purposes of this review are to highlight the many challenges to diagnosing, treating, and preventing C. difficile infection in humans, and to stress that collaboration between human and veterinary researchers is needed to control this pathogen.

Acute effects of short-term subcutaneous terbutaline on theophylline disposition.

Theophylline and subcutaneous terbutaline are frequently used concurrently in the management of acute asthma. Recent evidence demonstrating a reduction in theophylline serum concentrations during concomitant oral terbutaline therapy prompted our evaluation of subcutaneous terbutaline's effect on theophylline pharmacokinetics. Using a randomized, placebo controlled, crossover design, the disposition of a single oral theophylline dose (7 mg/kg) was studied in eight healthy, adult males before and after repeated subcutaneous administration of terbutaline (0.25 mg). Two-way analysis of variance revealed no significant difference in elimination rate constant (ke), area under the concentration-time curve (AUC), or apparent oral clearance (CL/F) of theophylline following terbutaline administration. These results indicate that subcutaneous administration of terbutaline does not alter the pharmacokinetics of single, oral doses of theophylline in adults.

Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids.

A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a beta-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate. In a model system using pSP65 or pGEM4 plasmids and transcripts, biotinylated RNA probes allowed detection of 5 pg of DNA in 10(6) pg of exogenous nucleic acids in 1000 min. Signals generated in the system depended on input target length. A nucleic acid target of 25 bases was still detectable in the assay. Human immunodeficiency virus type 1 (HIV-1) DNA was amplified in the polymerase chain reaction with Taq polymerase and a set of primers for the pol gene, one of which contained T7 RNA polymerase promoter sequences. A HIV-RNA probe of 326 bases was transcribed with T7 RNA polymerase using polymerase chain reaction (PCR) amplified DNA as a template. The RNA probe of 326 bases performed as well as a RNA probe of 2588 bases for detection of a DNA segment of 355 bp. For detection of dilutions of HIV-1 with PCR, a set of primers (outer set) was used for amplification of HIV-1 DNA. In a separate reaction a set of primers nested between the first set generated through PCR an amplified DNA fragment with the T7 promoter. This fragment was transcribed for the synthesis of a biotinylated RNA probe. This probe could then be reacted with material amplified with the outer set of primers. Ten copies of HIV-DNA could be detected with this procedure.

Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay.

A sensitive and specific system for detection of amplified Chlamydia trachomatis DNA from cervical specimens by fluorometric quantitation in an enzyme immunoassay (EIA) format (polymerase chain reaction [PCR]-EIA) is described. The primers selected for PCR-amplified DNA were from the 15 serovars of C. trachomatis and two strains of Chlamydia pneumoniae (TWAR). One strain of Chlamydia psittaci (Borg) was not amplified. One hundred four previously cultured cervical specimens were evaluated. Forty-six culture-positive specimens containing from 1+ to 4+ inclusion bodies were all positive by PCR-EIA. Of 58 culture-negative specimens, 2 were repeatedly positive and were nonreactive with control probes. This assay system represents a sensitive and specific combination of technologies for the quantitative detection of C. trachomatis DNA directly from a body fluid.

Evidence for a predominant proinflammatory conjunctival cytokine response in individuals with trachoma.

Immune responses to Chlamydia trachomatis infection in trachoma do not protect against reinfection or the development of scarring and blindness. In addition, the immunoregulatory contribution of cytokines to the development of conjunctival histopathology or protection is undefined. In this study, conjunctival cytokine mRNA transcripts were compared among subgroups of chlamydia infection status and ocular disease presentations of 50 individuals from an area where trachoma is endemic. There was a significant association of elevated interleukin (IL)-1beta, transforming growth factor beta1, and tumor necrosis factor alpha transcripts with infection, follicular inflammation, and scarring. Both gamma interferon (IFN-gamma) and IL-2 transcripts were significantly associated with infection; slightly elevated IL-2 levels were found in inflammatory disease. High IFN-gamma transcript levels were present with follicles and inflammatory disease and to a lesser extent with inflammatory scarring. The role of IFN-gamma in protection from infection or disease was not apparent from this study, since transcripts were frequently present in both chlamydial infection and disease. IL-12 (p40) transcripts were elevated in adults and children in association with follicular inflammation but not with scarring. IL-4, IL-5, and IL-10 transcripts were not detected in any samples. In conclusion, C. trachomatis infection stimulates local cytokines which favor a strong cell-mediated and proinflammatory response in both the early and later manifestations of trachoma. In addition, cytokine transcript levels that were associated with disease but no infection were characteristically lower overall than when chlamydia was present.

Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia.

The purpose of this study was to analyze hepatobiliary samples of patients with biliary atresia for rotavirus groups. A, B, and C, because group A rotavirus had been used to produce an animal model of the disease and group C rotavirus had been found in hepatobiliary samples from one group of patients. Biliary remnants and liver tissue from 10 biliary atresia and 14 control patients with other liver diseases were examined for rotavirus groups A, B, and C using nonisotopic, reverse transcriptase polymerase chain reaction enzyme immunoassay. Biliary atresia patients had a median age of 3 mo and were from a confined geographic area. Rotaviral stocks from groups A and C were used as polymerase chain reaction-positive controls. The limits of detection for rotaviral RNA from these two groups were respectively, 5 plaque-forming units and 50 tissue culture infectious doses (ID50). Tissue culture was 100-fold less sensitive for groups A and C than the polymerase chain reaction. The nested nonisotopic probes hybridized in solution only with their homologous target DNAs as determined by the enzyme immunoassay, or by Southern blot hybridization. Although it was possible to detect mRNA from a beta-actin housekeeping gene in all of the hepatobiliary samples, no evidence of rotaviral RNA was found in either the biliary atresia or the negative control group. In conclusion, rotavirus is not a common viral etiology of biliary atresia.