Brucellosis in pastoral and confined livestock: prevention and vaccination.

The traditional lifestyle and beliefs of pastoralists and small-scale farmers with confined livestock, together with certain farming environments, create favourable conditions for the spread and transmission of brucellosis. The risks associated with these practices are difficult to control because of a lack of alternatives and simple and/or affordable solutions. Brucellosis affects the health and productivity of livestock as well as that of their owners and caretakers and can have a deep economic impact. The control of brucellosis is likely to be cost effective. Good quantitative information on brucellosis in livestock and the human population is essential for demonstrating the benefits of intervention. Effective vaccines for the control of brucellosis in cattle and small ruminants are available and cheap, and in high-risk areas there is an urgent need to start large-scale vaccination programmes. Risks for the spread and transmission of brucellosis, such as the migration of herds with frequent contacts with other herds at common feeding grounds and near water sources, are inherent in the way of life of pastoralists. Such risks may need to be accepted when developing a control programme. Thus, the control of brucellosis by vaccination is expected to be more effective for confined livestock. Essential to the success of mass vaccination in controlling brucellosis is achieving a high degree of protection of adult livestock in a very short period and vaccinating young stock before natural infection can occur. To reduce the risk of transmission of infection from neighbouring areas where animals are not vaccinated, a region-wide approach is important. Because shepherds and farmers may have very little knowledge of infectious diseases and the consequences of infection, providing disease information and education is important to help them understand the need for control measures. Public health services can also assist in encouraging acceptance of control programmes in livestock by creating awareness of brucellosis as a human disease. To reduce costs, brucellosis control programmes can be combined with other veterinary or public health activities or interventions. An up-to-date livestock census and an effective surveillance system are crucial for the control of brucellosis, as the disease may quickly re-emerge from remaining foci of infection. Although test and slaughter may be an option for the management of remaining or re-emerging foci of infection, such a strategy is frequently not an option because of the cost.

Microbiological quality and antibiotic residues in informally marketed raw cow milk within the coastal savannah zone of Ghana.

OBJECTIVES: To investigate the microbiological quality and the presence of antibiotic residues in raw cow milk and in some indigenous milk products produced and marketed by the informal sector in the coastal savannah zone of Ghana. METHODS: Milk samples were aseptically collected from 224 kraals and samples of 26 indigenous milk products were purchased from processors and retailers. Total plate counts, total coliform counts and the presence of Escherichia coli and E. coli O157:H7 were determined in all 250 samples. Milk samples were also tested for antibiotic residues. RESULTS: Total plate counts exceeded 105 CFU/ml in 45.2% of the samples while coliforms exceeded 10³ CFU/ml in 66.0% and E. coli was detected in 11.2%. E. coli was present in raw cow milk but not in the indigenous products and all E. coli isolates were negative for E. coli O157:H7. Antibiotic residues were detected in 3.1% of the raw cow milk samples. CONCLUSION: Bulk milk contains unacceptable levels of hygiene indicators and antibiotic residues and is a potential source of milk-borne infections. The detection of E. coli and antibiotic residues raises public health concerns about the safety of fresh unpasteurized cow milk in the coastal savannah zone of Ghana and calls for improved farm hygiene, the need for milk pasteurization and the sensible use of antibiotics in the milk industry.

Safety and immunogenicity of subcutaneous or intramuscular administration of a monovalent inactivated vaccine against Leptospira interrogans serogroup Icterohaemorrhagiae in healthy volunteers.

The safety and immunogenicity of a monovalent inactivated vaccine against Leptospira interrogans serogroup Icterohaemorrhagiae was evaluated in 84 volunteers according to the route of administration, i.e., subcutaneous (SC) or intramuscular (IM), in a double-blind randomised trial. The volunteers were randomised into four groups: SC vaccine; IM vaccine; SC placebo; and IM placebo. Primary vaccination comprised two injections on day 0 and day 14, with a booster after 6 months. A second booster was given 30 months after primary vaccination. Local reactions within 1 h of injections were rare, with no difference between vaccine groups. Local reactions within 3 h were more frequent after the second, third and fourth SC injections than after IM injections. Systemic reactions never occurred within 1 h of vaccination and were rare within 3 days; the rates were comparable for the different vaccine groups. Evolution of the antibody responses, as assessed by microscopic agglutination tests and specific IgG and IgM ELISAs, were similar for both injection routes. IgG seroconversion rates after the first booster were 97% (95% CI 80-100%) for the SC vaccine group, and 96% (95% CI 80-100%) for the IM vaccine group, and both reached 100% for IgG after the second booster. The safety and immunogenicity of the anti-leptospiral vaccine were both good. Monitoring of antibody levels established that a booster dose triggered a strong antibody response in fully vaccinated subjects at 30 months after primary vaccination.

Molecular cloning of gene sequences regulated during squamous differentiation of tracheal epithelial cells and controlled by retinoic acid.

A cDNA library was constructed from polyadenylated RNA present in squamous differentiated rabbit tracheal epithelial cells. Screening of the cDNA library was aimed at identifying RNAs that were abundant in squamous cells and expressed at low levels in undifferentiated cells. Two different recombinants were obtained containing inserts, 0.86 and 0.77 kilobases (kb) in size, that hybridized to mRNAs 1.0 and 1.25 kb in length. These RNAs were present at approximately 50-fold higher levels in squamous cells than in proliferative or confluent retinoic acid-treated cells. The increase in the levels of the 1.0- and 1.25-kb RNAs correlated closely with the onset of squamous differentiation and was not related to induction of terminal cell division. Treatment of rabbit tracheal epithelial cells with transforming growth factor beta, which induces squamous differentiation in these cells, also resulted in elevated levels of the 1.0- and 1.25-kb RNAs. The increased levels of these RNAs in squamous cells appeared to a large extent to be regulated at a posttranscriptional level. Retinoic acid not only inhibited the increase in the levels of the 1.0- and 1.25-kb RNAs but also reversed the expression of these RNAs in squamous cells. These results suggest that retinoic acid affects, directly or indirectly, molecular events that induce alterations in the posttranscriptional processing of the transcripts corresponding to the 1.0- and 1.25-kb RNAs.

Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro.

1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. The products of synthesis are short (Mr less than 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate/urea, guanidinium . HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase.

Terminal alpha (1 leads to 4)-linked N-acetylglucosamine: a characteristic constituent of duodenal-gland mucous glycoproteins in rat and pig. A high-resolution 1H-NMR study.

The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract was examined for species- and tissue-specificity. To this purpose, oligosaccharides were released from purified glycoprotein preparations of rat and pig gastric, duodenal-gland and small-intestinal mucus, by alkaline borohydride reductive cleavage. Based on the results of 500-MHz 1H-NMR spectroscopy and of sugar analysis of the total oligosaccharide fractions, terminal GlcNAc, alpha (1 leads to 4)-linked to galactose, appears to be a characteristic constituent of duodenal-gland oligosaccharides. Similarly, NeuAc in alpha (2 leads to 3)-linkage to galactose turns out to be a typical constituent of small-intestinal mucous glycoproteins. In general, glycoproteins from gastric mucus possess larger and more-branched carbohydrate chains than those from duodenal-gland and small-intestinal mucus. Comparing rat and pig, oligosaccharide structures for corresponding tissues are less complex for the former. After fractionation, the rat duodenal-gland oligosaccharides could be characterized by application of 1H-NMR spectroscopy as being branched tetra- up to hexa-saccharide chains, all sharing the italicized trisaccharide element. The chains exhibit microheterogeneity as to the termination by fucose in alpha (1 leads to 2)- or by GlcNAc in alpha (1 leads to 4)-linkage to galactose. The following structures can be proposed for the most abundant rat duodenal-gland oligosaccharides: (table; see text).

Leptospirosis: an emerging health problem in Thailand.

Leptospirosis is an emerging health problem in Thailand, with dramatic increases in reported incidence since 1996. The annual number of reported leptospirosis cases increased from 398 cases in 1996 to 14,285 cases in 2000. In 2001, 2002, and 2003, the number of reported cases decreased, but still remained high at 10,217, 6,864, and 4,958 cases, respectively. The epidemiological characteristics of leptospirosis in Thailand include a peak incidence in September and October in association with the rainy season. A vast majority of the cases (90%) were reported in the Northeast region. The case fatality rate was as high as 4.4%, having a predominant association with male farmers aged 15 to 45 years. Outpatient cases were approximately 9 times more common than admitted cases, with an apparent recent shift in the pattern of infecting serovars among reservoir animals and humans.

High frequency of detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in biopsies from malignant and premalignant skin lesions from renal transplant recipients.

Based on immunologic and epidemiologic data, it is plausible that skin cancer in renal transplant recipients is associated with human papillomaviruses (HPV). At present, conflicting evidence exists concerning the presence of HPV DNA in these cancers. We recently described a nested polymerase chain reaction method that enables the detection of all previously isolated epidermodysplasia verruciformis (EV)-associated HPVs. We now describe the detection of EV-associated HPV DNA in 49 (80%) of 61 biopsies from squamous cell carcinomas, in four (50%) of eight basal cell carcinomas, in 14 (93%) of 15 actinic keratoses, in two (40%) of five cases of Bowen's disease, and in four (57%) of seven keratoacanthomas. HPV DNA typing revealed that all detected HPV types belonged to the EV-associated HPV types. A wide spectrum of EV-associated HPVs was found, including six putative new HPV types. In a high percentage of the lesions more than one HPV type was detected. We often found the same HPV types in different skin biopsies from both malignant and premalignant lesions from the same patient. The high frequency of detection of EV-associated HPV types in biopsies from malignant and premalignant lesions is in agreement with the hypothesis that EV-associated HPVs are involved in the pathogenesis of skin cancer in renal transplant recipients.

No evidence of known types of human papillomavirus in squamous cell cancer of the oesophagus in a low-risk area. Rotterdam Oesophageal Tumour Study Group.

Controversial results regarding the presence and role of human papillomavirus in the development of oesophageal squamous cell carcinoma have been published. We used multiple broad-spectrum polymerase chain reactions to identify HPV DNA in oesophageal carcinomas from a low-incidence area. Paraffin embedded- and snap-frozen specimens from oesophageal cancer tissues of 63 patients were examined with a PCR technique with several primer pairs, capable of detecting most known HPV types. In none of the oesophagus cancer tissues could HPV DNA be detected. The role of HPV in this type of carcinoma in a low incidence area remains unclear.

Risk factors for HPV detection in archival Pap smears. A population-based study from Greenland and Denmark.

The most important risk factor for cervical cancer is genital infection with certain types of human papillomavirus (HPV). The presence of HPV was studied in archival smears from a random sample of women living in Greenland (GW) and Denmark (DW) having, respectively, a high risk and an intermediate risk for cervical cancer. Risk factors were also examined of the original 126 Danish and 129 Greenlandic archived smears collected during October and November 1988. 125 were located from each country including all abnormal smears. HPV DNA was isolated from the smears and detected by means of a consensus polymerase chain reaction (PCR) detecting a broad spectrum of genital HPV types. HPV was detected in all the abnormal smears and in 22 and 33% respectively of the cytological normal smears from DW and GW. Risk of HPV was significantly higher in smears from women who started sexual life relatively recently (respectively, < or = 4 and < or = 6 years ago in DW and GW) compared with > or = 10 years ago (adjusted prevalence-OR: 9.3; 95% CI: 2.2-39.2 in DW and 5.9; 95% CI: 1.4-25.3 in GW). Among other important risk factors were age in both areas, lifetime number of sex partners and current smoking in DW and ever and gonorrhoea in GW. This study confirms the usefulness of the method as all abnormal smears were positive and, furthermore, the predictors for HPV presence in the normal smears corroborate with those found in recent studies of HPV in fresh cervical swabs. Thus, this method can be useful for large-scale epidemiological studies of HPV DNA in already sampled material.

A simple dipstick assay for the detection of antibodies to phenolic glycolipid-I of Mycobacterium leprae.

Among the many reported applications of the detection of antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae, in particular, the use of seroprevalence as an indicator of the magnitude of the leprosy problem may turn out to be very useful in leprosy control programs. An operational function of serology within the leprosy control services requires a simple test system. We have developed a simple dipstick assay for the detection of antibodies to PGL-I and compared its performance with that of an ELISA. A high degree of agreement (97.2%) was observed between the ELISA and the dipstick assay when tested on 435 sera; the agreement beyond chance (Kappa value) was 0.92. No significant difference was found between the dipstick assay and the ELISA when seropositivity rates obtained in groups of leprosy patients, household contacts, and controls were compared. The interpretation of the dipstick results as positive or negative was unequivocal, as illustrated by the high agreement between different persons reading the test (Kappa values > 0.88). Storage of the only reagents required, the dipsticks and the stabilized detection reagent, up to three weeks under tropical conditions of high temperatures, high humidity, and exposure to light, did not influence the results of the assay. The dipstick assay described here is an easy-to-perform method for the detection of IgM antibodies to PGL-I of M. leprae; it does not require any special equipment and the highly stable reagents make the test robust and suitable for use in tropical countries. An internal control validates the performance of the assay. This dipstick assay may be the method of choice for epidemiologic mapping of leprosy.

Increase of leptospirosis in dengue-negative patients after a hurricane in Puerto Rico in 1996 [correction of 1966].

Leptospirosis has rarely been reported in Puerto Rico, although in the period from 1948 to 1952, 208 cases of leptospirosis and an island-wide seroprevalence of antibody to Leptospira of 14% were documented. In Puerto Rico in October 1996, following rainfall and a period of flooding generated by Hurricane Hortense, serum specimens of 4 patients with suspected dengue fever that were negative for dengue tested positive for Leptospira-specific IgM antibodies in a dipstick assay. Subsequently, we used an island-wide dengue laboratory-based surveillance system to determine the increase in leptospirosis after hurricane-generated floods. All anti-dengue IgM-negative patients (n = 142) with disease onset from August 8 to October 6, 1996 from prehurricane and posthurricane groups were investigated for leptospirosis. Laboratory-confirmed leptospirosis cases were defined as microscopic agglutination test titers > or = 1 :400 to 1 or more serovars, or positive immunohistochemistry in autopsy tissues. Four (6%) of 72 prehurricane and 17 (24%) of 70 posthurricane patients had laboratory-confirmed cases of leptospirosis (relative risk [RR] = 4.4, 95% confidence interval [CI] = 1.6-12.4). The mean age of case-patients was 34 years (range = 13-64). Eighteen (86%) of 21 confirmed case-patients were males, including one patient who died (31 years old). Patients were located in 18 (38%) of 48 municipalities that submitted serum samples. Clinical features significantly associated with leptospirosis were eye pain (RR = 1.5, 95% CI = 1.3-1.9), joint pain (RR = 1.4, 95% CI = 1.1-1.6), diarrhea (RR = 1.7, 95% CI = 1.2-2.5), and jaundice (RR = 3.3, 95% CI = 1.5-7.2). This study demonstrates the utility of a dengue laboratory-based surveillance system for the detection of an increase of leptospirosis, which most likely would have gone unrecognized. Leptospirosis is treatable with antibacterial agents; knowledge of this diagnosis may significantly reduce morbidity and mortality.

Evaluation of three methods to measure anti-Brucella IgM antibodies and interference of IgA in the interpretation of mercaptan-based tests.

The results of a dipstick assay for the detection of immunoglobulin M (IgM) to Brucella smooth lipopolysaccharide (S-LPS) correlated with those of an enzyme-linked immunosorbent assay (ELISA) for IgM and of the serum agglutination test (SAT) performed with and without dithiothreitol. Two sera which were dithiothreitol-sensitive and were dipstick negative were shown to contain specific IgA. The dipstick assay is recommended as a simple method for detecting specific IgM antibodies in acute-phase brucellosis patients.

Detection of cutaneous and genital HPV types in clinical samples by PCR using consensus primers.

Two sets of consensus PCR primers consisting of a common 3' primer CP-I and two 5'-primers, CP-IIG (primer set A) and CP-IIS (primer set B), in the E1 open reading frame of the human papillomavirus (HPV) genome are presented. These two primer sets enabled the detection of a 188 base pair (bp) fragment of HPV 1, 2, 3, 4, 5, 6b, 7, 8, 9, 10a, 11, 12, 14a, 16, 17, 18, 19, 20, 21, 22, 24, 25, 31, 33, 36, 37, 38, 39 and 46. HPV types 15, 23, 49 and 50 were poorly amplified and HPV type 41 was not amplified. The method is suitable for the detection of HPV DNA sequences in clinical samples of both cervical and cutaneous lesions.

Application of the NASBA nucleic acid amplification method for the detection of human papillomavirus type 16 E6-E7 transcripts.

Using a human papillomavirus type 16 (HPV-16) E6-E7 specific primer set in a nucleic acid sequence-based amplification (NASBA) reaction, detection of HPV-16 transcripts was accomplished in a single enzymatic reaction at 41 degrees C. The NASBA reaction product was visualized either by Northern bolt analysis with an HPV-16 E6-E7-specific 32P-labelled oligonucleotide probe or by a non-radioactive enzyme-linked gel assay (ELGA). In combination with a rapid nucleic acid extraction procedure this method appears to be very suitable for the sensitive and specific detection of HPV-16 transcripts on small amounts of HPV-16-expressing cells of various sources, including cervical smears.

Clinical utility of a dipstick assay in patients with brucellosis: correlation with the period of evolution of the disease.

To examine the clinical utility of a dipstick assay for the detection of Brucella-specific IgM antibodies, and the correlation with the evolution of the disease. Twenty-six patients who were admitted to the General Hospital of Albacete (Spain) over a 2-year period and diagnosed with brucellosis were included in the study. One hundred and twenty-five serum samples collected at the time of diagnosis and at intervals during and after treatment were tested by the Coombs test, the standard seroagglutination test (SAT), the SAT in the presence of dithiothreitol (DTT-SAT), and a dipstick assay for the detection of Brucella-specific immunoglobulin M (IgM) antibodies. The sensitivity of the dipstick assay at the moment of the diagnosis was similar to that of the SAT (62% and 73%, respectively), somewhat higher than that of the DTT-SAT (50%), and lower than that of the Coombs test (100%). Patients with a negative dipstick test at the moment of diagnosis displayed a period of evolution of the disease longer than that of the dipstick-positive patients. After the beginning of therapy, the detection rate of the dipstick assay decreased faster than those of the SAT, the DTT-SAT, and the Coombs test. Thirty days after the start of therapy, the detection rate of the dipstick assay had decreased to 7%, whereas that of the SAT and DTT-SAT was 46%, and that of the Coombs test was still 92%. The dipstick assay could be used as a rapid diagnostic test for patients in the early stages of illness. Patients with a long period of illness will probably have a negative dipstick test, and could be diagnosed with the aid of the Coombs test and classical clinical findings.

Nursing home reimbursement and the allocation of rehabilitation therapy resources.

Most public funding methods for long-term care do not adequately match payment rates with patient need for services. Case-mix payment systems are designed to encourage a more efficient and equitable allocation of limited health care resources. Even nursing home case-mix payment systems, however, do not currently provide the proper incentives to match rehabilitation therapy resources to a patient's needs. We were able to determine by a review of over 8,500 patients in 65 nursing homes that certain diagnoses, partial dependence in activities of daily living (ADLs), clear mental status, and improving medical status are associated with the provision of rehabilitation services to nursing home residents. These patient characteristics are clinically reasonable predictors of the need for therapy and should be considered for use in nursing home case-mix reimbursement systems. Primary payment source also was associated with the provision of rehabilitation services even after taking into account significant patient characteristics. It is unclear how much of the variation in service use across payers is due to differences in patient need as opposed to differences in the financial incentives associated with current payment methods.

Graduate medical education consortia: expectations and experiences.

Graduate medical education (GME) consortia are formal associations of medical schools, teaching hospitals, and other organizations involved in residency training, with central support, direction, and coordination allowing members to function collectively. GME consortia are not well documented in the literature, although they are included in many GME reform proposals. Proponents expect consortia to improve the organizational structure and governance of residency training programs and, through local coordination and possibly the allocation of resources, to help members meet national workforce goals regarding both numbers and practice locations for generalist and specialist physicians. The authors contrast the expectations of educators and policymakers for the future performance of GME consortia with the experiences of 36 functioning consortia, gathered in a 1993 national survey conducted by the Maine Medical Center and the Association of American Medical Colleges. Respondents account for nearly 10% of all residency training programs and 17% of all residents nationwide. The participating consortia differ markedly in their structures, functions, and other features, although nearly 85% have been convened by medical schools and closely affiliated hospitals. Fewer than half function with governing boards; 40% offer a single integrated residency training program in each discipline; and 58% allocate resources. A common concern of consortium members was physician distribution by specialty, but only 31% identified "increasing the number of generalist physicians" as a major accomplishment to date. To improve their performance, consortium participants call for greater commitment and leadership from the medical school and university, better definition of the community's GME needs, more information about the flow of GME funds locally, greater authority to allocate resources, and incentives for shifting allegiances from individual institutions to the consortium community as a whole.

Incentives in case-mix measures for long-term care.

Several States now use patient-based payments for skilled nursing facilities and intermediate care facilities; others are in the process of developing case-mix systems. The Health Care Financing Administration is working under congressional mandate to develop a prospective case-mix system for Medicare payments to skilled nursing facilities. If new payment methods follow the existing pattern, they will be based not on the patient's clinical characteristics but rather on a mixture of clinical characteristics and services delivered. As a result, innate incentives are contained in data collection systems which are cost-increasing at best and dangerous at worst. A preferable approach would be to develop payment schemes based on the patient's degree of dependence.

Variation in resource use within diagnosis-related groups: the severity issue.

Several authors have suggested that diagnosis-related groups (DRG's) make inadequate allowance for the severity of illness. Before modifications of DRG's are developed, the sources of within-group variation must be precisely defined; not all variation is attributable to the severity of illness. The limitations of the Uniform Hospital Discharge Data Set (UHDDS), of the International Classification of Diseases, Ninth Revision, Clinical Modification coding system and of the original rules of DRG construction must be evaluated and, if necessary, corrected before new approaches to groupings are considered. The most promising potential modifications of existing groups and weights are those that make use of the UHDDS, or of the UHDDS plus additional diagnoses and procedures. The addition of entirely new data elements to the discharge abstract and the pricing process should be considered only as a last resort.