A critical review of prescriptions in internal medicine.

A study was undertaken to assess the prescribing trends in inpatients of the Internal Medicine Department of Government General Hospital which is attached to Guntur Medical College, Guntur. Prescriptions for 250 patients were audited under various heads like details of drugs used, generic versus trade name prescribing, dosage, dosage form, dose interval, drug of choice and duration of treatment. Among 250 prescriptions audited, 7 percent used generic names, 22 percent trade names, and 71 percent combinations of both generic and trade names. Dosages and dosage intervals were appropriate. In 75 percent of the audited cases, the duration of treatment was appropriate; 25 percent did not specify the duration. The choice of drugs was proper in 84 percent and inappropriate in 16 percent of cases. Certain deficiencies in the prescribing trends are analysed.


Atypical presentations of falciparum malaria.

To identify the uncommon presentations of falciparum malaria in an endemic area and to assess the outcome of treatment, a study was carried out on 35 proved cases whose clinical presentations were either dominated by features other than fever or the history of fever was totally absent. Both urban and rural patients were included. Seventeen cases (48.3%) presented with features of cerebral malaria. Acute abdomen, urticaria, and unexplained shock were the other atypical presentations. Five cases (14.3%) of cerebral malaria died. We conclude that awareness of atypical presentations is important to detect cases of falciparum malaria in an endemic area. Intravenous quinine may need to be given promptly even when cerebral malaria is diagnosed empirically.


Study of foetal antigen in haematological malignancy.

Forty patients with different varieties of leukaemia and lymphoma were studied before and after therapy. Red cells and lymphocytes from each patient were tested for foetal antigen by lectin-agglutination test. The antigen was detectable on red cells in all untreated cases, the highest titre being found in chronic myeloid leukaemia. The titre showed significant reduction after treatment in all cases. We conclude that foetal antigen on red cells is a useful diagnostic aid in haematological malignancy and is a good indicator of the outcome of therapy.


Diagnostic evaluation of ascitic adenosine deaminase activity in tubercular peritonitis.

Activity of adenosine deaminase (ADA) in serum and peritoneal fluid was studied prospectively in 24 aetiologically proved cases of ascites and 10 age-matched controls. Patients were divided into 3 groups according to causes of ascites, viz. malignant ascites (11), tubercular peritonitis (7) and cirrhosis of liver (6). Serum ADA values and peritoneal: serum ADA ratio did not show any consistent pattern in any group. But in patients with tubercular peritonitis ADA activity in ascitic fluid was significantly higher (P < .001) than in the other groups. An ascitic ADA level of 30 units/L had a sensitivity of 100% and specificity of 94.1% for tubercular peritonitis. These findings suggest that the ascitic fluid ADA activity is useful for the diagnosis of tubercular peritonitis; this method is simple and least invasive.


Prevalence of hepatitis surface antigen among rural population of Loni area in Ahmednagar district of Western Maharashtra.

Three hundred and fifty three subjects among the rural population of Loni area admitted in the hospital, and 188 medical staff members working in the Rural Medical College and Hospital, Loni, were screened for the presence of HBsAg. Reveresed passive haemagglutination assay was used for screening; it showed an HBsAg positivity rate of 21.8% and 15.8% among hepatitis and non hepatitis cases respectively, and 1.2%, 0% and 4.2% among medical students, doctors and nursing staff respectively. A high HBsAg positivity rate has been observed in the rural population of Loni area.


Pneumocystis carinii pneumonia in untreated acute non-lymphoblastic leukaemia.

Mixed opportunistic infection with Pneumocystis carinii and Candida prior to cytotoxic therapy in a young male diagnosed as having acute non-lymphoblastic leukaemia resulted in early catastrophe. The role of awareness of this complication and its prompt management is discussed.


Primary sclerosing cholangitis.

A case of primary sclerosing cholangitis is reported. There was no history of choledocholithiasis, operation or traumatic biliary injury.


Decreased pulmonary barotrauma with the use of volumetric diffusive respiration in pediatric patients with burns: the 1992 Moyer Award.

Pulmonary barotrauma is a frequent, life-threatening complication in the pediatric patient who is treated with mechanical ventilation. The volumetric diffusive respiration (VDR) ventilator, which employs a high-frequency progressive accumulation of subtidal volume breaths in a pressure-limited format with a percussive waveform, is capable of providing adequate gas exchange at lower airway pressures; this theoretically decreases the incidence of pulmonary barotrauma compared with conventional mechanical ventilation (CV). The incidence of pulmonary barotrauma since 1988 was evaluated in pediatric patients with burns who were younger than 2 years of age. Twenty-four patients who were treated with only CV were compared with 15 patients who were treated with only VDR. Pulmonary barotrauma was defined as the development of pneumothorax, pneumomediastinum, pneumopericardium, or pneumoperitoneum. There were no significant differences between CV-treated and VDR-treated groups (mean +/- SEM) in the patient characteristics of age (15.9 +/- 1.3 months vs 16.6 +/- 1.8 months), weight (11.2 +/- 0.5 kg vs 12.5 +/- 0.7 kg), percent total body surface burn (46.2% +/- 4.9% vs 55.6% +/- 6.2%), percent full-thickness burn (38.1% +/- 5.3% vs 50.0% +/- 6.6%), inhalation injury (40% vs 60%), or total number of days that mechanical ventilation was required (18.2 +/- 4.2 days vs 22.4 +/- 5.9 days); although these parameters show a slightly more severe degree of injury in the VDR-treated group. There was a reduction in the incidence of pulmonary barotrauma when VDR was used.(ABSTRACT TRUNCATED AT 250 WORDS)


Clinical trials of a living dermal tissue replacement placed beneath meshed, split-thickness skin grafts on excised burn wounds.

We evaluated the ability of Dermagraft (Advanced Tissue Sciences, La Jolla, Calif.), a living tissue analog that is composed of human neonatal fibroblasts, which are grown on a polyglactin acid Vicryl mesh (Ethicon Inc., Somerville, N.J.), to function as a dermal replacement when placed beneath meshed, expanded split-thickness skin grafts (MESTSGs). Full-thickness burn wounds in 17 patients with burns (mean age, 31 years; range, 6 to 69 years; mean burn size, 23.8% total body surface area) were excised to subcutaneous fat (nine patients), to fascia (three patients), or to a combination of deep dermis and fat (five patients). Dermagraft was placed over the experimental sites, which were then covered with MESTSGs. Paired control sites on each patient received MESTSGs only. The results showed that "take" of MESTSGs on control sites was slightly better than take on experimental sites that contained the Dermagraft; however, the differences were not statistically significant. Mesh interstices epithelialized over the surface of the full-thickness wound (control sites) or over the surface of Dermagraft (experimental sites). Wound biopsy specimens demonstrated no evidence of rejection of the cultured allogeneic fibroblasts and minimal inflammatory reaction to the Vicryl fibers. Evidence of continuous basement membrane formation at the epithelial-Dermagraft junction, which was identified by immunohistochemical staining for laminin and type IV collagen, was seen by day 14 beneath the healed epithelium in the skin graft interstices. The Vicryl fibers were hydrolyzed in the wound over a 2-to-4-week period, although some expulsion of fibers occurred as the healing epithelium advanced to close the MESTSG interstices. Elastic fibers were not seen in neodermal tissue in either control or experimental wounds at periods of up to 1 year after grafting. Further trials with this living tissue replacement are in progress.


Efficacy of leupeptin, superoxide dismutase, and verapamil in modulating delayed reperfusion damage after burn injury.

Reperfusion damage has been identified as an important factor in multiorgan failure after severe burn injury. We wondered if leupeptin, a protease inhibitor, superoxide dismutase (SOD), a scavenger of free oxygen radicals, or verapamil, a calcium antagonist, would protect the cellular energy metabolism when they were given with fluid resuscitation that was delayed 6 hours after a severe burn injury. Fifty male rats weighing 280 to 300 gm received a 50% third-degree scald burn. Ten of these received fluid resuscitation at 30 minutes and 1 1/2 hours after injury, and 40 received delayed fluid resuscitation at 6 and 7 hours after injury. Thirty of these 40 rats were given leupeptin (n = 10), SOD (n = 10), or verapamil (n = 10). Heart, liver, and kidney tissue samples were obtained 8 hours after injury; adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate were measured; and the energy charge potential was calculated. Tissue water content (TWC) in lung and skeletal muscle was also determined. The adenine nucleotide pool and the energy charge potential in heart, liver, and kidney tissue were all significantly decreased (p < 0.01) in rats receiving delayed fluid resuscitation compared with those receiving early resuscitation. Leupeptin was effective in protecting the heart against reperfusion damage, and verapamil and leupeptin showed some efficacy in protecting kidney tissue. Liver tissue, however, showed no protective response with therapy. TWC was significantly decreased (p < 0.01) in skeletal muscle with SOD treatment, and though all treatments appeared to keep lung water content reduced, none was significant at p < 0.01. We thus conclude that both the decreases in heart and kidney adenine nucleotides and the increase in TWC that are caused by delayed fluid resuscitation can be attenuated with appropriate pharmacologic agents.


Pseudomonas aeruginosa exotoxin A: its role in retardation of wound healing: the 1992 Lindberg Award.

Bacterial concentrations greater than 10(5) colony-forming units/gm of tissue prevent wound healing. However, it has not been determined whether it is the number of bacteria or a toxin produced by these organisms that impedes the wound healing process. Pseudomonas aeruginosa (PSAR), a burn wound pathogen, produces a dermonecrotic toxin, exotoxin A. Studies have indicated a role for exotoxin A in the pathogenicity of PSAR. We investigated the role of exotoxin A in the retardation of contraction. Acute granulating wounds were created on 90 Sprague-Dawley rats. The animals were equally divided into six groups and were treated topically as follows: group 1, sham: no infection, no treatment; group 2, exotoxin A; group 3, exotoxin A and antiexotoxin; group 4, autoclaved PSAR 10(6); group 5, 10(6) viable PSAR inoculated in the wound; group 6, 10(6) viable PSAR and antiexotoxin. Wound contraction was measured with the use of planimetry twice a week. Serial biopsies were performed on all wounds. Contraction rates revealed significantly (p < 0.05) retarded closure in the animals treated with exotoxin A and in the viable PSAR group when compared with the rates of the noninfected control groups. Animals treated with exotoxin A plus antiexotoxin A and those treated with live PSAR and antiexotoxin showed contraction rates identical to the control groups. These data suggest that exotoxin A in PSAR infections retards wound healing and that neutralization of the toxin restores the normal healing process.


Effect of indomethacin and Impact liquid diet administration on inflammatory mediator production by murine splenocytes after burn injury.

Production of prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) by various cells is increased after injury, and these mediators are implicated in the downregulation of immune responses. We attempted to modulate the production of immune suppressive mediators by inhibiting prostaglandin production in a murine model that had a burn covering 25% of the total body surface area. Two treatments were performed. One was the intraperitoneal administration of indomethacin after burn injury at 2 mg/kg day for 10 days. The control group received saline solution injections. The other treatment was the ad libitum administration of a commercial diet after burn injury for 10 days. This diet contained mixed fats, including omega-3 and omega-6 fatty acids. The control group received standard mouse food. On days 1, 5, and 10 after burn injury, spleens were removed aseptically, and splenocyte cultures were established and stimulated with phytohemagglutinin. TNF-alpha and PGE2 concentrations were determined in culture supernatants at 48 hours with the use of commercial enzyme-linked immunosorbent assay kits. Splenocytes from burned animals produced elevated levels of both mediators in culture supernatants, reaching significant levels of TNF-alpha on day 10 after burn injury and of PGE2 on days 5 and 10 after burn injury. Neither the administration of indomethacin for 10 days nor the administration of the commercial diet for 10 days decreased production of these mediators in culture. However, cells in culture may escape the in vivo regulating effects of biologic modifying agents.


Modification of calcium flux of twitch skeletal muscle in mice subjected to 20% body surface area burn.

One systemic effect of burn trauma is skeletal muscle weakness. This is the result of changes in second messenger systems involving calcium (Ca2+). Kinetic analysis of Ca2+ from cellular compartments of skeletal muscle of mice that were subjected to small burn injury (20% body surface area) was performed. Muscles of the burn group showed an increased 45Ca2+ uptake maxima compared with those of time-matched control groups. Also, 45Ca2+ efflux analysis showed a lack of differentiation between electrically stimulated tissues and nonstimulated tissues that was easily observed in time-matched control groups. This lack of differentiation indicated changes in intracellular compartmentation. It can be speculated that burn trauma may have a regulatory role in the excitation-contraction coupling mechanism by altering myoplasmic levels of Ca2+ even before skeletal muscle dysfunction occurs. These findings suggest that agents capable of controlling myoplasmic Ca2+ levels may aid in alleviating chronic skeletal muscle dysfunction in patients with burns.


How long do we need to give antioxidant therapy during resuscitation when its administration is delayed for two hours?

Twenty-four guinea pigs with third-degree burns over 70% of the body surface area were divided equally into four groups. All animals received Ringer's lactate (R/L) beginning 30 minutes after burn injury. Group 1 received R/L without vitamin C beginning 2 hours after burn injury. Groups 2, 3, and 4 received R/L with vitamin C until 4, 8, and 24 hours after burn injury, respectively. Beginning 3 1/2 hours after burn injury the hourly fluid volume was reduced to 25% of the Parkland formula calculation. The hourly sodium and fluid intake in each group was the same. Groups 1 and 2 demonstrated higher hematocrit and lower cardiac output values as compared with those values for group 3, indicating hypovolemia and hemoconcentration in these groups. Group 3 showed hematocrit and cardiac output values equivalent to those values for group 4. We conclude that high-dose vitamin C must be given until 8 hours after burn injury to maintain adequate hemodynamic stability in the presence of a reduced resuscitation fluid volume.


A distraction technique for control of burn pain.

Research has indicated that analgesics alone do not adequately relieve pain for 75% of patients with burns. The purpose of this study was to determine the effects of a distraction therapy, in which videos were used in combination with administration of analgesics, on intensity and quality of pain and on levels of anxiety in adults during burn dressing changes. The sample consisted of 17 patients who were randomly assigned to the treatment or the control group. The treatment group viewed video programs that were composed of scenic beauty accompanied by music. Each was asked to score his or her present pain intensity and pain rating index with the McGill questionnaire and anxiety with the Spielberger questionnaire before and after the dressing change. A nested general linear model using the "F" test in multiple regression analysis was adjusted for age, percent partial-thickness burn, and choice of topical agent demonstrated that the use of videos during the dressing changes significantly reduced pain and anxiety: present pain intensity (F = 8.69; p = 0.01), pain rating index (F = 5.57; p = 0.03), anxiety (F = 9.10; p = 0.01). It is recommended that the use of pain medication be augmented by use of videos during burn dressing changes.


Micrografts: the "super" expansion graft.

A novel technique for producing micronized skin grafts that was introduced in a paper presented at the 1990 ABA meeting was evaluated to quantify maximum expansion. Twenty Sprague-Dawley rats were divided into two groups representing 10:1 and 25:1 expanded micrograft ratios, respectively. Grafted sites in both groups were shown to heal better than those of the control group, and both grafted groups showed comparable healing at day 10.


Effects of high-dose vitamin C administration on postburn microvascular fluid and protein flux.

The effects of vitamin C treatment (14 mg/kg/hr) on burn injury were evaluated in the hind paws of 12 mongrel dogs. A lymph duct above one hind paw of each dog was cannulated. Hourly lymph flow rates (QL) and plasma and lymph total protein concentrations were measured before the burn injury and for 6 hours after the burn injury. Data from 24 paws were divided into four groups: nonburn without treatment, nonburn with treatment, burn without treatment, and burn with treatment. The nonburn groups showed no significant differences in QL or in total protein flux. In the burn groups the postburn hourly QL increased by sevenfold in the nontreatment group and only by threefold in the treatment group, whereas the postburn hourly total protein flux increased by fifteenfold and fivefold, respectively. We conclude that administration of high-dose vitamin C reduces early postburn microvascular leakage of fluid and protein.


NSAID-induced gastroenteropathy: a biochemical dissection.

The most widely accepted view is that damage to the mucosa sets the stage for lesions related to NSAIDs. An alternative hypothesis is that NSAID-induced damage to GI microvasculature is the primary pathologic event.


Use of morphine sulfate (MS Contin) in patients with burns: a pilot study.

Morphine sulfate (MS Contin), a proven analgesic in the treatment of cancer pain and chronic benign pain, seems to be a good analgesic for the treatment of burn pain. MS Contin is morphine sulfate incorporated in a wax cellulose matrix delivery system. This wax cellulose delivery system gives MS Contin its duration of action. Ten patients were enrolled in an open-labeled, nonrandomized study. The study was designed to examine the analgesic efficacy of MS Contin in the burn population. Each patient remained in the study for 6 days. The efficacy of the analgesic regimen was subjectively measured by the visual pain scale. The MS Contin group was retrospectively compared with a group of patients who were given continuous intravenous infusions of morphine. The two groups were matched according to age, burn size, surgical procedures, and hospital stay. The analgesic qualities of MS Contin were comparable to those of continuous intravenous morphine sulfate infusions. MS Contin is a possible candidate for the treatment of patients with burn pain because of its analgesic qualities, oral dosing, and duration of action.


Inhalation injury: a decade without progress.

Despite major advances in the management of patients with critical burn injuries, inhalation injury continues to be a major determinant of death resulting from burn injuries. Two cohort groups of victims with burn and inhalation injuries, separated by a decade, were retrospectively reviewed in an effort to determine the impact of newer treatment modalities. Patients were categorized as being at "high" or "low" risk on the basis of primary and secondary diagnostic criteria. Despite a statistically significant decrease in the percent of total body surface injury, no change in mortality rate was noted between the two groups. The advent of sophisticated diagnostic and management techniques does not appear to have decreased the mortality rate associated with inhalation injury.


Updating upper extremity temporary prosthesis: thermoplastics.

Since 1989 amputees with upper-extremity burns have been fitted with a temporary prosthesis fabricated from low-temperature thermoplastic. Before 1989 conventional temporary prostheses were fabricated with plaster. The use of the thermoplastic material has produced a lightweight, cost-effective, modular system. No patients exhibited skin breakdown with the thermoplastic material. It appears that thermoplastics may be the next major breakthrough in terms of a design for a temporary upper-extremity prosthesis.


Mixing inpatient with outpatient care: establishing an outpatient clinic on a burn unit.

Outpatient care of patients with burns is an important aspect of a total health care plan. Changes in the health care system, which focuses on cost containment, force reevaluation of the methods used for delivery of high-tech care, particularly in areas such as burn care. Great advances that have taken place over the past decade in the field of burn care have enabled health care providers to treat more patients with burns as outpatients. Those who are specially trained in burn care continue to be the optimal caregivers. The appropriate facilities, spray tables, hydrotherapy, and dressing rooms in which patients with burns are treated are equally important and must be adapted to meet the needs of patients who are ambulatory. The goals of an outpatient burn clinic should be to provide daily wound care and patient education to prevent unnecessary admissions and to promote early discharge for hospitalized patients. Nurses trained in burn care are the optimal providers of ambulatory burn care; therefore the clinic location should be where the caregivers are available. Several obstacles needed to be overcome before an outpatient clinic could be established on the burn unit itself. Wound care is now provided by burn unit nurses, which leads to better results and more consistent follow-up. Patient satisfaction is increased, patient teaching is provided by experienced staff, unnecessary admissions are prevented, and patients are able to be discharged from the hospital earlier or to be followed as outpatients even if surgery is eventually required.


Burn center reimbursement analysis.

If burn centers are to survive in the current fixed-payment environment, managers and physicians must be knowledgeable of the parameters and regulators of reimbursement. The purpose of this study was to assess the reimbursement status of a burn center in a private, not-for-profit facility. The analysis included both internal and external constraints imposed on the hospital. Data from fiscal year 1989-90 were used to identify the most frequently coded burn diagnosis-related groups and third-party payers. Actual reimbursement rates per diagnosis-related group were examined, and the average charge per patient discharge was compared with reimbursement per patient. Total billed charges versus received revenue for Medicaid patients were explored. Problems and solutions were identified and discussed after completion of the assessment.


Color pressure garments versus traditional beige pressure garments: perceptions from the public.

Social acceptance of patients with burns may be affected by the color of the pressure garment they wear. The purpose of this study was to ask students (n = 712), health care providers (n = 354), and the public (n = 250) their perceptions of beige versus color pressure garments. It was assumed that beige pressure garments have negative connotations and that colored pressure garments have positive connotations. Data were collected from subjects after they viewed beige, pink, and blue pressure garments. Items on the instrument contained bipolar word pairs rated on a 5-point scale; a rating of 1 indicated positive feelings, and a rating of 5 indicated negative feelings. Cronbach's alpha reliability for the instrument was 0.85, indicating a high internal consistency among items. Paired t test indicated a significant difference between the subjects' perceptions of the beige garments versus their perceptions of the color garments (t = -44.82, p = 0.000) among all subjects (n = 1259). Results indicate that positive feelings toward color garments may lead to social acceptance of the patient with burns, which in turn could increase the patient's self-esteem.


When is enough enough? Ethical dilemmas on the burn unit.

Modern burn care often leads to the dilemma of what should or should not be done for patients with clinical deterioration and organ system failure who fail to respond to therapy. The questions are, "When is enough enough?" and "Who decides?" We have developed a structured conference to address these issues and to help us decide whether to recommend continued invasive diagnostic and therapeutic intervention or to allow the patient to "die with dignity." This conference can be requested by any member of the burn team who feels uncomfortable with what is being done for and/or to a patient. It is a meeting of the entire team, and its purpose is to discern the judgment of the group. When the consensus decision is to forego additional therapy, the decision is then presented to the patient (if he or she is able to understand and respond) and to the patient's family. The decision made by the group removes the responsibility of any individual from making a stressful decision if the patient's condition deteriorates abruptly. Patients who accept this decision exhibit a peaceful calm that invariably reaffirms the group dynamics. The family often experiences a great deal of relief, because they are not forced to make the decision even though they wanted it made. Inviting nurses to be active participants in the decision process builds their personal and professional self-esteem and binds the team members into a more tightly knit community. The attending staff may perceive this process as an abdication of responsibility; however, in our experience the consensus conference has led to a conviction that the wisdom of the team is always best.


Liposomes: realizing their promise.

Thirty years of study have proved that these artificial organelles are versatile tools for learning about the cell membrane. Researchers are now actively investigating their potential for some interesting practical uses as vehicles for gene transfer, as artificial erythrocyte surrogates, as a lubricant for degenerated joint surfaces, and most imminently as "targeted carriers" of pharmacologic agents.


Determination of fatty acid and triacylglycerol composition of human adipose tissue.

The fatty acid composition of adipose tissue was studied in a population in western Andalusia with a diet in which the fat contribution is mainly from olive oil. The lipid composition of adipose tissue, including the fatty acid composition of triacylglycerols, was examined by capillary gas chromatography. Thirty-five peaks were resolved, ranging in chain length from 12 to 24 carbon atoms, including geometric and positional isomers. The major triacylglycerol was POO, followed by PLO and OOO.


Application of gas chromatography-mass spectrometry with selected ion monitoring to the urinanalysis of 4-pyridoxic acid.

An analytical protocol has been developed for the analysis of urinary 4-pyridoxic acid (4-PA) by gas chromatography-mass spectrometry (GC-MS) for use in metabolic studies. Aliquots of urine were deproteinised and fractionated by isocratic reversed-phase high-performance liquid chromatography. The eluent fraction containing the 4-PA was collected, freeze-dried and silylated using N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. Derivatisation produced the mono-tert.-butyldimethylsilyl derivative of 4-PA lactone. This derivative was readily amenable to GC-MS analysis in the electron ionisation (70 eV) mode, yielding a prominent fragment ion at m/z 222 ([M-57]+; base peak). A heavy isotope-labelled derivative of pyridoxine [dideuteriated pyridoxine; 3-hydroxy-4-(hydroxymethyl)-5-[hydroxymethyl-2H2]-2-methylpyridine] has been synthesised and is being employed to determine the kinetics of labelling of the body pools of vitamin B6. Kinetic measurements are based on the determination of the relative proportions of metabolically produced deuterium-labelled and non-labelled 4-PA in urine, obtained from stable isotope ratios determined by low-resolution selected ion monitoring using a bench-top quadrupole GC-MS system.


Characterisation of oligosaccharides from a glycoprotein variant of human serum albumin (albumin Casebrook) using high-performance anion-exchange chromatography and nuclear magnetic resonance spectroscopy.

The characterisation of oligosaccharides present on albumin Casebrook, a glycoprotein variant of human serum albumin, which contains an N-linked oligosaccharide at an attachment site formed by a point mutation of 494 Asp-->Asn, is described. The monosaccharide compositional analysis of purified glycopeptides suggested the presence of complex biantennary carbohydrate structures. The oligosaccharides which were released by N-glycosidase-F appeared to be a single molecular species according to their retention on high-performance anion-exchange chromatography. The structure of the oligosaccharide was suggested by sequential exoglycosidase digestions and confirmed by proton nuclear magnetic resonance spectroscopy. It was concluded that the oligosaccharides were essentially homogeneous and consisted of an alpha(2-6)-desialylated complex biantennary glycan.


Gas chromatographic-mass spectrometric method for routine monitoring of 5-fluorouracil in plasma of patients receiving low-level protracted infusions.

A gas chromatographic-mass spectrometric (GC-MS) method is described which quantitates 5-fluorouracil (5-FU) plasma levels ranging from 0.5 to 50 ng/ml. The analysis uses two internal standards, 1,3-[15N2]-5-fluorouracil and 5-chlorouracil. Extraction and derivatization of the pyrimidine bases were accomplished in a single step using acetonitrile. Compounds were analyzed as their 1,3-dipentafluorobenzyl derivatives by electron-impact MS, and the GC-MS analysis was automated with respect to sample injection and data reduction. Stability of the analysis was demonstrated by continuous unattended analysis of 5-FU in human plasma for periods of up to three days with no deterioration of the quantitative results. The method is applicable to quantitating 5-FU plasma levels in patients receiving protracted infusions of the drug for colorectal cancer or other malignancies.


Identification of metabolites of halofantrine, a new candidate anti-malarial drug, by gas chromatography-mass spectrometry.

Two previously unknown metabolites of halofantrine, a candidate anti-malarial drug, have been isolated by thin-layer chromatography from the plasma of dogs administered a single oral dose of 60 mg/kg. Their identifies were investigated after trimethylsilylation by gas chromatography-mass spectrometry under electron-impact and negative-ion chemical ionization conditions. The structural assignment was further confirmed by using a combination of elemental composition analysis of all the isotope peaks at low mass resolution and isotope pattern matching. These two metabolites were formed by modification of the dibutylaminopropyl side-chain of the parent compound involving deamination and oxidation or reduction.


Comparison of high-performance liquid chromatography and high-performance capillary electrophoresis for the determination of cicletanine in plasma.

A sensitive and selective high-performance capillary electrophoresis (HPCE) procedure was developed for the determination of total cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using sodium dodecyl sulphate in the run buffers and ultraviolet detection. The concentrations of cicletanine obtained by this method were compared with those obtained by a high-performance liquid chromatographic (HPLC) method used routinely. The within-run precision of the methods, expressed as relative standard deviation, ranged from 1.6 to 7.8% for HPLC and from 6.4 to 11.1% for HPCE. Both methods showed an adequate level of accuracy; the relative errors ranged from 0.02 to 3.25% for HPLC and from 0.21 to 2.90% for HPCE. The HPCE method required less than half the time taken by the HPLC method, making HPCE a useful alternative technique for the routine determination of cicletanine in plasma. Both methods were used to follow the time course of total cicletanine in human plasma after a single oral therapeutic dose of the drug.


Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection.

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid-liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50-2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.


Determination of zolpidem, a new sleep-inducing agent, and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans.

For the determination of zolpidem, a new sleep inducer, and its metabolites in human plasma and urine, three methods were developed that are suitable for pharmacokinetics, drug metabolism and overdosing investigations. The methods used for pharmacokinetic and drug metabolism studies are based on column-switching high-performance liquid chromatography; they do not require any sample manipulation because the plasma or diluted urine is injected into a pre-column where clean-up and preconcentration take place. The analytes are transferred by valve-switching to the C18 analytical column for chromatography. To investigate overdose cases, urine samples only are used: the method is simple, because the diluted urine can be injected directly into the analytical column (phenyl type). This allows the identification and quantification of the principal urinary metabolite of zolpidem, the unchanged drug being practically undetectable. All the methods use fluorescence detection, which affords high sensitivity and selectivity. It is necessary to use a method capable of the determination of metabolites even if these are apparently pharmacologically inactive, because in different physiopathological populations the qualitative and quantitative metabolic profiles of zolpidem could be different. The method designed for the investigation of (accidental or deliberate) overdose cases is, as required on such occasions, simple and rapid, with good selectivity with respect to commonly prescribed psychotropic drugs.


High-performance liquid chromatographic determination of ambroxol in human plasma.

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid-liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.


Coupling of microdialysis with capillary electrophoresis: a new approach to the study of drug transfer between two compartments of the body in freely moving rats.

The feasibility of in vivo microdialysis and capillary electrophoresis for pharmacokinetic studies was tested. Microdialysis probes were inserted in the jugular vein and brain of rats. After an intraperitoneal injection of phenobarbital, microdialysis was performed in the brain and the blood simultaneously in each rat under freely moving conditions. Capillary electrophoresis with ultraviolet absorption was used to measure phenobarbital in blood and brain dialysates. The time course of phenobarbital in the blood and in the extracellular space of the brain was followed. The results demonstrated that microdialysis can be used for pharmacokinetic studies in freely moving animals. Capillary electrophoresis has the potential to improve the time resolution of microdialysis. Other advantages of microdialysis and capillary electrophoresis for pharmacokinetic studies are discussed.


Fluorimetric detection of serum corticosterone using high-performance liquid chromatography.

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.


Determination of urinary 3-methylhistidine by high-performance liquid chromatography with o-phthaldialdehyde precolumn derivatization.

A high-performance liquid chromatographic method to measure the concentration of 3-methylhistidine in human urine with o-phthaldialdehyde precolumn derivatization is described. A clear separation of the 3-methylhistidine derivative from other derivatives in urine was achieved within 25 min. The linearity, recovery and coefficients of variation were determined. The method was successfully applied to clinical assays and used for diagnostic purposes.


Assay of timolol in human plasma using gas chromatography with electron-capture detection.

A simple and sensitive gas chromatographic method has been developed for the determination of timolol in plasma using electron-capture detection and propranolol as internal standard. Timolol was extracted using butyl chloride and derivatized using trifluoroacetic anhydride in butyl acetate. The lower detection limit for the assay was found to be 1 ng/ml from 1 ml of plasma. Extracted standards gave within-day precision of 12.55, 9.68 and 3.78% for 1, 20 and 100 ng/ml plasma samples, respectively. A recovery of at least 80% of timolol was found using the extraction method described. The assay was used in a randomized cross-over bioequivalence trial using an oral administration of 20 mg of timolol. Pharmacokinetic parameters compare favourably with other literature values.


Analysis of 5-fluorouracil in plasma and urine by high-performance liquid chromatography.

A relatively simple, sensitive and rapid high-performance liquid chromatographic method is described for measuring the anticancer drug 5-fluorouracil (5-FU) in human plasma and urine. The procedure includes liquid-liquid extraction using ethyl acetate-methanol (95:5) and preparative column chromatography to separate 5-FU from constituents normally occurring in these biological samples. The columns contained a specially modified form of diatomaceous earth, which requires no pre-conditioning washes. Reversed-phase high-performance liquid chromatography was performed on a C18 column (70 mm x 4.6 mm I.D.) with a mobile phase of water-methanol (95:5) and ultraviolet detection (268 nm). The overall recovery from plasma and urine was 91 and 94%, respectively, at the concentration of 50 ng/ml. The determination limit of the assay for 5-FU was 10 ng/ml of plasma and urine. Concentrations of 5-FU between 10 and 500 ng/ml were measured in plasma and urine with a relative standard deviation of 6.8%. In order to evaluate the procedure, plasma and urine samples from three patients treated with 5-FU by continuous intravenous perfusion, were investigated.


High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine.

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C18 solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5.10(-4) M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.


High-performance liquid chromatographic determination of pilocarpine in plasma.

A high-performance liquid chromatographic procedure requiring neither derivatization nor complex sample work-up is reported for reproducibly and sensitively determining pilocarpine in plasma. Following stabilization of pilocarpine against in vitro hydrolysis using sodium fluoride, plasma samples were extracted and the extracts chromatographed on a 5-microns, low-carbon-load (6%) C18 reversed-phase column. The assay was linear between 10 and 300 ng/ml (r = 0.998). It had sufficient sensitivity to quantitate pilocarpine at concentrations as low as 10 ng/ml (signal-to-noise ratio > or = 4) using a 500-microliters sample. The assay appears to be the first published specifically for plasma determinations and has proven capable of supporting pharmacokinetics studies of pilocarpine disposition in the anesthetized dog.


Determination of zopiclone in plasma using column liquid chromatography with ultraviolet detection.

A reversed-phase liquid chromatographic method with ultraviolet detection for the determination of zopiclone in plasma is described. It is rapid, sensitive, reproducible and linear over a wide range. The method was used to study plasma zopiclone concentrations in a case of acute intoxication after oral ingestion of 300 mg of the drug. The plasma level was 1600 ng/ml 4.5 h after the dose and the elimination half-life was 3.5 h.


Determination of remoxipride in human plasma and urine by reversed-phase ion-pair high-performance liquid chromatography.

A sensitive method is described for the measurement of remoxipride in human plasma and urine. Remoxipride and its internal standard are extracted from plasma or urine at pH 12 with a mixture of hexane and methyl tert.-butyl ether. After washing the organic phase with base, the compounds are extracted into acid and analyzed on a C18 column with ultraviolet detection at 214 nm. The mobile phase is composed of acetonitrile and aqueous buffer (sodium perchlorate and phosphoric acid, pH 1.7). The limits of reliable quantitation for remoxipride are 12.5 and 50 ng/ml for plasma and urine, respectively. The run times are 6 min for plasma and 3 min for urine. The method has been successfully used to assay remoxipride clinical study samples. This mobile phase has also been successfully applied to the analysis of other basic drugs such as cimetidine, codeine, diltiazem and quinidine with minor modifications.


Evaluation of a high-performance thin-layer chromatographic technique for the determination of salbutamol serum levels in clinical trials.

Salbutamol concentrations were determined by high-performance thin-layer chromatography in the sera of two sets of ten volunteers at hourly intervals for 6 h after taking one 8-mg slow-release tablet. The influence of time lapse in processing of serum samples, i.e. centrifugation, extraction and chromatography, was studied. A statistical significant instability of salbutamol in the sera of patients was found which was not present in standard drug-free serum samples spiked with salbutamol and used for construction of standard curves.


Determination of tetracycline antibiotics by reversed-phase high-performance liquid chromatography with fluorescence detection.

A highly sensitive method for the determination of tetracycline antibiotics (TCs) using reversed-phase high-performance liquid chromatography with fluorescence detection is presented. This method was based on the use of disodium ethylenediaminetetraacetate (EDTA) and calcium chloride as fluorescence-increasing reagents in the mobile phase. The concentrations of each reagent in the mobile phase greatly influenced the fluorescence intensity of TCs. When the concentration of EDTA and calcium chloride were 25 and 35 mM, respectively, and the pH of the mobile phase was 6.5, the maximum fluorescence intensity was obtained. The column temperature hardly influenced the fluorescence intensity. At 3.75 ng of TCs injected, the precision (relative standard deviation) ranged from 1.12 to 2.20%. In the range 0.075-37.5 ng for tetracycline and oxytetracycline and 0.225-37.5 ng for chlortetracycline, a linear response was observed. The detection limits of this method were 49-190 pg for three different TCs. The proposed method was applied to the determination of one of the TCs in pharmaceuticals by the internal standard method using other TCs as internal standards and was also applied to determination of TCs added to fish tissue.


Separation of the enantiomers of some potassium channel activators using an alpha 1-acid glycoprotein column.

The separations of the enantiomers of some 3,4-dihydro-2,2'-dimethyl-2H-1-benzopyrans and a related tetrahydronaphthalene on alpha 1-acid glycoprotein (Chiral-AGP) are presented, together with the results from an investigation of the effects of organic modifier and pH on the separations achieved. The general utility of Chiral-AGP in separating the enantiomers of compounds from this class of antihypertensive agents is demonstrated in this paper.


Analysis of legume oligosaccharides by high-resolution gas chromatography.

The suitability of high-resolution gas chromatography (HGRC) for the analysis of the raffinose family oligosaccharides (raffinose, stachyose, verbascose) was investigated. Aqueous methanol (80%) extracts of pea flour were dried and derivatized with either trimethylimidazole or N-methyl-bis(trifluoroacetamide). Separation of the sugar derivatives was achieved utilizing a 10-m DB5-60W capillary column. The effects of carrier gas (He) flow-rate and split ratio on resolution and reproducibility were studied. HRGC analysis was characterized by excellent resolution and satisfactory reproducibility, and proved to be a rapid, sensitive method for quantitation of oligosaccharides in pea flours.


Efficient enantioselective separation and determination of trace impurities in secondary amino acids (i.e., imino acids).

An R-(-)-1-(1-naphthyl)ethyl carbamoylated-beta-cyclodextrin bonded phase in conjunction with a nonaqueous polar mobile phase was used for the highly selective enantioseparation of a number of secondary amino acids after their pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC). Under the conditions employed, the FMOC reagent served to "lock" the imino acid into their existing conformation thereby preventing the possibility of racemization. Furthermore, it served to increase the sensitivity to the point that trace level enantiomeric impurities were easily detected. Compared with separations that use traditional reversed-phase solvents, this method showed several advantages: higher selectivity towards the imino acid enantiomers investigated, shorter analysis times, faster equilibration of the column, more stable baseline and more sensitive fluorescence detection. The detection limits for FMOC derivatives of proline, trans-4-hydroxyproline, cis-4-hydroxyproline, pyroglutamic acid, 3,4-dehydroproline, thiaproline, penicillamine acetone adduct and pipecolic acid are in the low femtomole range. The method was used for evaluation of enantioselectivity of a number of "optically pure" commercial imino acid standards. Enantiomeric impurities as low as 0.0001% (1 ppm) can be determined in some cases. High precision determination of trace levels of D-imino acids in the presence of large amounts of corresponding (opposite) L enantiomer at 1, 0.1, 0.01% and below are demonstrated.


Determination of diaminopimelic acid in biological materials using high-performance liquid chromatography.

A method for the determination of diaminopimelic acid (DAPA) concentrations in feeds and rumen digesta by reversed-phase high-performance liquid chromatography using precolumn derivatization with o-phthaldialdehyde and fluorimetric detection was developed. Samples were oxidized and hydrolysed prior to analyses by HPLC. Hydrogen peroxide and formic acid were used for oxidation; hydrolyses were performed using 3 M hydrochloric acid under vacuum at 120 degrees C for 17 h. Oxidation allowed more space for DAPA-OPA peak elution and hydrochloric acid hydrolysis reduced sample clean-up and extended the column life. Hydrolysates were diluted, adjusted to pH 7 and filtered. A Beckman Model 507 autosampler with a precolumn derivatization cassette was used for the derivatization process and fluorimetric detection was used to measure the OPA derivatives. Samples were prepared in order to have on-column DAPA concentrations in the range 10-100 pmol. The relative recovery of the standard solutions added to the feed samples ranged from 98.4 to 102.8 %. The reproducibility of the method was evaluated by the analysis of eight alfalfa hay samples and eight alfalfa hay samples incubated in the rumen for 48 h and they yielded relative standard deviations of 2.04 % and 2.02%, respectively.


Thermal desorption cold trap-injection in high-resolution gas chromatography: multivariate optimization of experimental conditions.

In studies of low concentrations of volatile compounds in air, the method of adsorption on porous polymers and determination by thermal desorption cold trap-injection high-resolution gas chromatography is finding increasing application. Factors considered important for injection and chromatographic separation of volatile compounds by this method were investigated with the use of multivariate techniques. For the amount injected on to the chromatographic column, the factors of main importance were found to be the temperature of the injection block, the thickness of the internal coating of the cold trap and the flow-rate. Strong interaction effects were noted. For the sharpness of the chromatographic peaks, the flow-rate was the most important factor.


Differential diagnosis of HTLV-I and HTLV-II infections by restriction enzyme analysis of 'nested' PCR products.

A 'nested' polymerase chain reaction (PCR) assay is described which is capable of detecting single copies of human T-cell lymphotropic virus (HTLV) in genomic DNA extracted from peripheral blood mononuclear cells (PBMCs). A single set of 'nested' oligonucleotide primers, based on the highly conserved tax/rex region of the viral genome, was able to detect both HTLV-I and HTLV-II proviral sequences in clinical samples of diverse geographical origins, from the United States, Great Britain, Japan, the Caribbean, Italy, Greece, Iraq and West Africa. Rapid discrimination between HTLV-I and HTLV-II infections was achieved by restriction enzyme analysis of unpurified second-round PCR products, even in those cases in which serological assays had failed to provide a definitive result. Over a 2-year period, a total of 53 HTLV infections (37 HTLV-I and 16 HTLV-II) were identified by this technique and complete concordance with serological typing, available in 41 cases, was observed.


Enzyme-linked immunosorbent assay for measurement of IgG antibody to Molluscum contagiosum virus and investigation of the serological relationship of the molecular types.

An ELISA was developed for measuring levels of IgG antibodies to Molluscum contagiosum virus (MCV) using DNA typed purified virus as antigen. The assay was found to be specific and sensitive for antibodies to MCV as well as being economical in its use of antigen. A close relationship was found between antibody levels to the MCV molecular types 1, 1v and 2 by cross-testing sera on plates coated with the different molecular types of the virus as antigen (P < 0.001).


Geographical clusters of dengue virus type 2 isolates based on analysis of infected cell RNA by the chemical cleavage at mismatch method.

Genetic variation in 12 strains of dengue virus type 2, isolated from several epidemic areas in different years, was studied by chemical cleavage at mismatched cytosine in DNA:RNA heteroduplexes. End-labelled cDNA probes derived from the E and NS2A genes of the New Guinea C strain were hybridized to total RNA extracted from cells infected by individual isolates. Following modification of mismatched cytosine by hydroxylamine and nucleic acid strand cleavage by piperidine, the resulting fragments of radiolabelled probe were analysed by electrophoresis and autoradiography. The patterns of bands generated corresponded to the geographical groupings of the isolates. Thus this method is suitable in epidemiological studies for rapidly surveying a large number of isolates for genetic variation in a particular gene of interest.


Directed and divided attention in Alzheimer's disease: impairment in shifting of attention to global and local stimuli.

This study investigated the relative performance of Alzheimer's disease (AD) patients and normal controls on directed and divided attention reaction time (RT) tasks that involved the use of global-local stimuli (e.g., a large '1' made from small '2s'). Relative to normals, AD patients displayed disproportionately greater impairment on the divided attention task compared to the directed attention task. On the divided attention task, when the target remained at the same global-local level across consecutive trials, the AD patients displayed a greater facilitation effect than did the controls when responding to the second stimulus. However, when the target changed levels across consecutive trials (i.e., from the global to the local, or vice versa) the AD patients' RTs to the second stimulus were disproportionately slower than were the controls' RTs. These results demonstrated that AD patients are impaired in disengaging and shifting attention across levels of perceptual organization within the same stimulus.


Neuropsychological functioning in adolescents with diabetes.

Investigated the relationship between disease variables, neuropsychological performance, and psychosocial status in adolescents with Insulin Dependent Diabetes Mellitus (IDDM). The study group consisted of 85 adolescents, aged 14 to 16.5 years who had been diabetic for longer than 12 months. Parameters of both recent and long-term metabolic control were determined, including diabetic incidents such as severe hypoglycemia or ketoacidosis. The mothers completed standardised measures of adolescent adjustment, and the adolescents provided self-reports of psychosocial status. Neuropsychological functioning was evaluated with standardised tests of verbal and nonverbal intelligence, memory and new learning, visuo-graphic skills, mental flexibility, and problem-solving ability. Using retrospective accounts of disease history, there was no relationship between neuropsychological functioning and variables such as age of onset, chronic poor control, or major metabolic crises. The findings emphasise the need for a long-term, prospective study of a cohort of diabetic children from the time of diagnosis to clarify causal relationships, if any, between illness variables, neuropsychological performance, and psychosocial factors.


Cognitive rehabilitation of the hemineglect disorder in chronic patients with unilateral right brain damage.

Thirteen patients with a stabilized hemineglect symptomatology due to right-hemisphere lesions were subjected to a rehabilitation training specifically aimed at reducing the scanning deficit. The training consisted of four procedures (visual-spatial scanning, reading and copying training, copying of line drawings on a dot matrix, and figure description) which lasted 40 sessions. By the end of therapy, the patients as a group showed significant improvements on several standard tests of hemineglect. The results on a Semi-structured Scale for the Functional Evaluation of Hemineglect pointed to the extension of exploratory improvements to situations similar to those of daily life. In contrast, patients improved very slightly on a variety of standard visual-spatial tests, indicating the specificity of training in reducing the scanning defect. Seven patients were examined at a follow-up several months after the end of therapy and appeared stable on both standard and functional tests of neglect.


Cognitive dysfunction after aneurysm of the anterior communicating artery.

The present study examined the nature of the amnestic syndrome following aneurysm of the anterior communicating artery (ACoA) in humans. Eleven ACoA and 13 subjects with intracranial hemorrhages (ICH) elsewhere in the brain were administered a battery of standard neuropsychological tests. The ACoA group performed significantly worse than the ICH controls on tests of delayed verbal memory and on the Wisconsin Card Sorting Test, despite significantly higher Full Scale IQ. No significant differences were observed between groups on tests of immediate recall, attention and concentration, and visuo-spatial functions, although the ACoA group tended to perform better on many of these tests. The results do not support the hypothesis that the cognitive impairments observed following ACoA aneurysm are the result of diffuse cortical damage. The role of specific anterior cerebral structures in defining the "ACOA syndrome" are discussed.


Factorial structure of the Waterloo Handedness Questionnaire for control and learning-disabled adults.

This study examined the factorial structure of the 32-item version of the Waterloo Handedness Questionnaire (Steenhuis & Bryden, 1989) with a sample of control (n = 325) and learning-disabled (LD) (n = 147) subjects. On the basis of Principal Components Analyses, we have replicated the work of Steenhuis and Bryden (1989) who suggested that hand preference factors for control subjects are multifactorial in nature and are related more to "skilled" and "less skilled" activities rather than on distal/proximal musculature. Further, we have found that the factor structure for hand preference in LD adults is somewhat different than of normally achieving adults. The primary difference occurred within Factor 2. Unlike control subjects, whose Factor 2 items were classified as "less skilled" and preference was less lateralized, items loading on this factor for LD subjects were classified as "skilled" and showed strongly lateralized responses for both left and right handers. Presumably, LD adults show a less marked distinction between "skilled" and "less skilled" unimanual motor activities than control subjects suggesting different degrees of manual laterality between the groups.


Evidence of presymptomatic cognitive decline in Huntington's disease.

Asymptomatic persons at risk for Huntington's disease (HD) (N = 28) were assessed with neuropsychological, psychiatric, and neurologic tests while undergoing genetic linkage studies to determine their probability of carrying the HD gene. Those participants who were subsequently identified as probable gene carriers did not differ on neurologic or psychiatric examination from those subsequently identified as probable noncarriers. Neuropsychological data are presented for a subset of participants free of other conditions (such as alcoholism) putting them at risk for cognitive deficits. Among these subjects, probable gene carriers were inferior to probable noncarriers on the neuropsychological battery as a whole and on several individual tests involving learning and memory. The results suggest the presence of cognitive decline prior to identifiable motor impairments in HD.


Multicenter evaluation of the AntigEnz Chlamydia enzyme immunoassay for diagnosis of Chlamydia trachomatis genital infection.

To evaluate the AntigEnz Chlamydia enzyme immunoassay (EIA) (Baxter/Bartels, Issaquah, Wash.), we studied 320 men and 1,209 women attending clinics for sexually transmitted diseases in Baltimore, San Francisco, and Seattle. At examination, two separate swabs were obtained from each patient, one for chlamydial culture and one for EIA. Cervical samples were collected from women, and urethral samples were collected from men. The prevalence of chlamydial infection by culture was 9% in Baltimore (n = 532), 11% in Seattle (n = 500), and 9% in San Francisco (n = 497). To resolve specimens with discrepant culture and EIA results, the EIA transport buffer was centrifuged and the resuspended pellet was stained by direct immunofluorescence to determine whether elementary bodies were present. Overall sensitivity of the AntigEnz Chlamydia assay compared with culture was 87% in men and 86% in women, and overall specificities were 94 and 97%, respectively. Differences between centers were seen, with sensitivities ranging from 76% among men and 79% among women in Seattle to 100% among men and 95% among women in Baltimore. With a true positive considered to be either a culture-positive or an EIA- and direct immunofluorescence-positive specimen, the revised sensitivity was 91% in men and 88% in women. Overall revised specificity was 99% in both men and women. We conclude that in this high-prevalence population, the sensitivity and specificity of this assay compare favorably with those of other noncultural antigen detection tests for the diagnosis of chlamydial genital infection.


Seroprevalence of immunoglobulin M (IgM) and IgG antibodies to polysaccharides of Streptococcus pneumoniae in different age groups of Ecuadorian and German children.

The age-specific prevalence of serum immunoglobulin M (IgM) antibody to capsular polysaccharides of Streptococcus pneumoniae, as detected by enzyme-linked immunosorbent assay, was studied in 1,301 Ecuadorian children enrolled in a national nutrition and health survey. This prevalence was 6% in infants < 6 months old and increased to 28% in children 6 to 11 months old, 49% in those 12 to 17 months old, and 58% in those 18 to 23 months old. About 80% of the 5-year-old children had this antibody. When tested separately against six different capsular polysaccharides, serum IgM antibody reacted with decreasing frequency with serotype 3, 8, 19, 6, 23, and 1 capsular polysaccharides. We did not observe a broadening of the antibody response with increasing age in the sense that more and more serotypes were recognized. A similar age-related prevalence was found for IgM antibody to the species-specific C-polysaccharide of S. pneumoniae and for IgG antibody to capsular polysaccharides of S. pneumoniae. A smaller German serum collection showed a comparable age-related prevalence of pneumococcus-specific serum IgG and IgM antibodies. The highest incidence of respiratory diseases was observed in 1- and 2-year-old Ecuadorian children. It thus seems that acquisition of serum antibody to S. pneumoniae reflects more the developmental maturation of an immune response than an actual exposure to different pneumococcal serotypes.


Detection of specific antibodies in gingival crevicular transudate by enzyme-linked immunosorbent assay for diagnosis of human immunodeficiency virus type 1 infection.

The purpose of this open and multicenter trial was to determine the usefulness of antibody detection by enzyme-linked immunosorbent assay (ELISA) in gingival crevicular transudate (GCT), which was collected with an investigational device (Orasure; Epitope, Beaverton, Oreg.), for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection and to compare it with antibody detection in serum. A total of 1,880 individuals were tested, as follows: 354 HIV-1-infected individuals (111 asymptomatics individuals and 243 individuals with AIDS), 46 individuals with autoimmune diseases (AD), 296 individuals with dental diseases, 42 individuals with other chronic diseases, and 1,142 healthy individuals. Sera from 356 individuals and GCT from 354 individuals were positive for HIV-1 antibodies. There were two false-negative gingival samples, one from an HIV-1-positive asymptomatic individual and one from a patient with AIDS. HIV-1 antibodies were unexpectedly detected in both serum and GCT of two individuals, one with dental disease and one with pulmonary tuberculosis. None of the sera or GCTs from healthy subjects or patients with AD were positive. Compared with the serum assay, the sensitivity, specificity, and positive and negative predictive values of the GCT assay were 99.5, 100, 100, and 99.9%, respectively. Of 355 paired serum-GCT samples that were HIV-1 positive by ELISA and that were tested by Western blot (immunoblot), all were positive for HIV-1 by using the U.S. Public Health Service interpretation criteria, while among gingival samples, 301 were positive, 52 were indeterminate, and 2 were negative. Of 82 negative paired samples selected at random, 80 were negative by Western blotting of serum and GCT and 2 were indeterminate by Western blotting of serum and negative by Western blotting of GCT (a healthy blood donor and a patient with dermatopolymyositis). Testing for HIV-1 antibodies in GCT is a simple and reliable screening procedure in populations with high and low prevalences of infection because of the high sensitivity and specificity of the method, and it offers improved safety for hospital personnel.


Whole-cell protein electrophoresis for typing Mycobacterium tuberculosis.

A method of discriminating between strains of Mycobacterium tuberculosis by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins combined with a sensitive silver stain is described. Thirty-five isolates of M. tuberculosis and five isolates from other species of Mycobacterium were examined, including serial isolates from the same patients and isolates from a small cluster of hospital cases. Different species of Mycobacterium were clearly distinguished, and within the species M. tuberculosis, different fingerprints were obtained, allowing discrimination of some strains from different patients. The reproducibility and discrimination of the technique are discussed.


Use of a recombinant 170-kilodalton surface antigen of Entamoeba histolytica for serodiagnosis of amebiasis and identification of immunodominant domains of the native molecule.

We expressed the gene that encodes one of the major surface antigens of Entamoeba histolytica, the 170-kDa protein (1,270 amino acids), as a glutathione S-transferase fusion protein containing amino acids 1 to 1202 (lacking the putative transmembrane and cytoplasmic regions) and as separate fusion proteins containing each of three major domains of the 170-kDa molecule. Lysates from bacteria induced to express one of these proteins were used as the target antigens in a Western blot (immunoblot) analysis to determine whether a recombinant 170-kDa antigen could serve as the basis for a serologic test used to detect invasive amebiasis and whether there are differences in humoral immunogenicity among the three major domains of the 170-kDa antigen. Among patients with invasive amebiasis from three major areas where the disease is endemic and two sites in the United States, 54 (90%) of 60 had antibodies to the recombinant 170-kDa protein. Among 37 patients from regions where the disease is endemic and 20 patients from the United States without amebic disease, 1 (2%) of 57 had antibodies to the recombinant 170-kDa protein. We found significant differences in seroreactivity to each of three major domains of the molecule among patients seropositive for the complete construct, ranging from 100% seroreactivity with the fusion protein containing the domain designated cysteine rich and 89% seropositivity with the fusion protein incorporating a portion of the region designated cysteine poor to only 9% seropositivity for the fusion protein containing the pseudorepeat domain. Our study indicates that a serologic test based on the recombinant 170-kDA antigen could serve as a highly sensitive and specific test for acute invasive amebiasis.


Evaluation of three immunoassays for detection of Chlamydia trachomatis in urine specimens from asymptomatic males.

The performances of three commercially available immunoassays (Chlamydiazyme/Antibody Blocking Assay [Abbott Diagnostics, Abbott Park, Ill.], IDEIA [Analytab Products, Plainview, N.Y.], and Microtrak EIA [Syva Co. Palo Alto, Calif.]) were evaluated for the detection of Chlamydia trachomatis in urine specimens from asymptomatic males. Assay results were compared with direct specimen immunofluorescence (DFA) analysis of urine sediment (Syva Microtrak; Syva Co.), which was chosen as the study confirmation assay. An overall Chlamydia prevalence of 7% (24 of 340) was found in our study population, with peak incidences occurring in the adolescent (8 of 93 specimens) and young adult (11 of 146 specimens) age groups. Sensitivity and specificity data among the Chlamydiazyme, IDEIA, and Microtrak enzyme immunoassays (EIAs) were determined to be 79.1 and 99%, 91.7 and 98%, and 95.8 and 99%, respectively. The Microtrak EIA and IDEIA products demonstrated sensitivities and specificities equal to or greater than those claimed for urine specimens. The diagnostic accuracies of these assays on asymptomatic subjects, along with the ease of this collection method, suggest a role for these products as screening tools. The sensitivity of the Chlamydiazyme assay was lower than that claimed previously in symptomatic patients, with 5 of 24 positive specimens demonstrating false-negative results. In those cases, centrifugation of the original immunoassay aliquot material and then DFA examination confirmed specimen positivity. Urine immunoassay screening in combination with DFA confirmation (which was chosen because it has antibody epitopic specificity different from that of the primary assay) provides a high degree of diagnostic precision. The use of noninvasive collection methods could result in greater testing compliance among asymptomatic males and, subsequently, could reduce the incidences of both symptomatic and silent chlamydial infections.


Scedosporium inflatum osteomyelitis in a dog.

Scedosporium inflatum Malloch et Salkin was found to cause osteomyelitis in a 6-year-old spayed female beagle. The previously healthy dog suddenly developed right-forelimb lameness. Bony changes consisting of proliferation with some lysis were noted on radiographic examinations. Microscopic observations of stained sections of tissue obtained by biopsy of the distal humerus revealed the presence of septate branching hyphae. Cultures inoculated with tissue from a later biopsy yielded a mold subsequently identified as S. inflatum. Tissue sections stained with specific Scedosporium fluorescent-antibody conjugate were positive, further substantiating the diagnosis. Although the dog was treated with oral itraconazole, no improvement in the animal's condition was noted, and it was euthanized. Autopsy revealed dissemination of the etiologic agent to the lungs.


Detection of Shigella dysenteriae type 1 and Shigella flexneri in feces by immunomagnetic isolation and polymerase chain reaction.

A combination of immunomagnetic separation (IMS) and a polymerase chain reaction (PCR) procedure was used for direct isolation and identification of Shigella dysenteriae type 1 and Shigella flexneri from feces. Immunomagnetic particles were coated with monoclonal antibody MASFB, which is specific for a common epitope of the O polysaccharides of S. dysenteriae type 1 and S. flexneri. Bacteria bound to the beads were boiled in water, and target DNA was amplified with a primer pair specific for a gene coded on the invasion-associated locus (ial) of the large virulence plasmid of all four Shigella spp. and enteroinvasive strains of Escherichia coli. A 320-bp DNA fragment was generated and detected by an alkaline phosphatase-conjugated probe. Nonviable cells were also captured and detected by this technique. The method is simple and fast (7 h) and has a detection limit of ca. 10 Shigella organisms per g in fecal samples. The combined IMS-PCR assay correctly identified all 57 samples carrying S. dysenteriae type 1 and 68 samples carrying S. flexneri from 238 fecal specimens and also permitted detection of 17 samples carrying Shigella spp. from 113 specimens from diarrheal patients in whom shigellae were not detected by conventional culture.


A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.

Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively.


Identification of meningococcal serosubtypes by polymerase chain reaction.

The polymerase chain reaction was used as the basis of a novel typing method for Neisseria meningitidis. Southern hybridization experiments demonstrated that it was possible to identify genes encoding different serological variants of the meningococcal class 1 outer membrane protein by probing with polymerase chain reaction products corresponding to known epitopes. A set of 14 defined variable regions was prepared in bacteriophage M13mp19 by the cloning of polymerase chain reaction products. The phage were dot blotted onto membrane filters, which were used as targets for hybridization of radiolabeled amplified class 1 outer membrane protein genes. Thus, the presence of many different subtype-specific epitopes could be investigated in one experiment. This technique was evaluated with a set of serological reference strains, mainly of serogroup B organisms, and provided an alternative, rapid, and comprehensive typing system that was capable of distinguishing known serosubtypes and also of defining currently untypeable strains independently of sodium dodecyl sulfate-polyacrylamide gel electrophoresis or serological analysis. An additional advantage of this technique was that in the case of an unknown serosubtype (i.e., one that did not hybridize with any of the known samples), the DNA amplified from the original sample could be used to determine the nucleotide sequence of the novel serosubtype and to clone the corresponding variable region into bacteriophage M13. It may be possible to develop this procedure for the diagnostic detection and typing of meningococci directly from clinical samples even when culture is not possible because of antibiotic treatment of an acute case.


Epidemiology of Tsutsugamushi disease in relation to the serotypes of Rickettsia tsutsugamushi isolated from patients, field mice, and unfed chiggers on the eastern slope of Mount Fuji, Shizuoka Prefecture, Japan.

A total of 59 strains of Rickettsia tsutsugamushi were isolated from patients (24 isolates), Apodemus speciosus mice (30 isolates), and unfed larvae of Leptotrombidium scutellare (2 isolates) and Leptotrombidium pallidum (3 isolates) in the Gotenba-Oyama District, Shizuoka Prefecture, Japan. All these isolates were classified into the three serotypes Karp, Kawasaki, and Kuroki based on reactivity with strain-specific monoclonal antibodies. Kawasaki- and Karp-type rickettsiae were isolated from L. scutellare and L. pallidum, respectively, and the geographic distribution of patients and rodents infected with these two types of rickettsiae coincided with the areas densely populated by the respective chiggers. From these results, we conclude that Kawasaki-type rickettsiae are transmitted by L. scutellare and Karp-type ones are transmitted by L. pallidum. Kawasaki-type rickettsial infections were prevalent in early autumn, and Karp-type infections showed a peak of occurrence in the late autumn, reflecting the seasonal fluctuations of L. scutellare and L. pallidum. Isolates of Kuroki-type rickettsiae were obtained only from four patients in October and November, and the relationship between this type of rickettsia and its vector species could not be fully defined.


Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction.

A rapid and sensitive polymerase chain reaction (PCR)-based assay for detection of Chlamydia trachomatis in cervical specimens is described. This assay consists of (i) sample preparation which avoids the use of heat, centrifugation, or organic extractions; (ii) rapid, two-temperature PCR amplification of C. trachomatis cryptic plasmid sequences; and (iii) capture and colorimetric detection of amplified DNA in microwell plates. PCR was compared with culture by using 503 cervical specimens. After resolution of discrepant specimens with a confirmatory PCR assay directed against the chlamydial major outer membrane protein gene, PCR had a sensitivity of 97% and a specificity of 99.7% while culture had a sensitivity of 85.7% and a specificity of 100%. In a separate study, PCR was compared with a direct specimen enzyme immunoassay (Chlamydiazyme; Abbott Diagnostics) by using 375 cervical specimens. After resolution of discrepant specimens, PCR had a sensitivity and specificity of 100%, while the enzyme immunoassay had a sensitivity of 58.8% and a specificity of 100%.


Polymerase chain reaction-based detection of Trypanosoma cruzi DNA in serum.

DNAs prepared from chagasic patients' sera were amplified by the polymerase chain reaction using oligonucleotide primers which anneal specifically to a highly repetitive sequence of Trypanosoma cruzi nuclear DNA. Samples from both acutely and chronically infected patients yielded positive results by this method. No significant difference was observed when either whole blood or serum samples of the patients were used. These results indicate that serum instead of whole-blood samples could be used for polymerase chain reaction-based detection of T. cruzi in field studies without the need of applying any special chemical treatment to the specimens. This would represent a considerable advantage due to the easier handling and transportation of serum as compared with whole-blood samples, especially in tropical climates.


Improved stool concentration procedure for detection of Cryptosporidium oocysts in fecal specimens.

Epidemiologic and laboratory data suggest that coprodiagnostic methods may fail to detect Cryptosporidium oocysts in stool specimens of infected patients. To improve the efficacy of stool concentration procedures, we modified different steps of the Formalin-ethyl acetate (FEA) stool concentration technique and evaluated these modifications by examining stool samples seeded with known numbers of Cryptosporidium oocysts. Because these modifications failed to improve oocyst detection, we developed a new stool concentration technique that includes FEA sedimentation followed by layering and flotation over hypertonic sodium chloride solution to separate parasites from stool debris. Compared with the standard FEA procedure, this technique improved Cryptosporidium oocyst detection. The sensitivities of the two concentration techniques were similar for diarrheal (watery) stool specimens (100% of watery stool specimens seeded with 5,000 oocysts per g of stool were identified as positive by the new technique, compared with 90% of stools processed by the standard FEA technique). However, the most significant improvement in diagnosis occurred with formed stool specimens that were not fatty; 70 to 90% of formed stool specimens seeded with 5,000 oocysts were identified as positive by the new technique, compared with 0% of specimens processed by the standard FEA technique. One hundred percent of formed specimens seeded with 10,000 oocysts were correctly diagnosed by using the new technique, while 0 to 60% of specimens processed by the standard FEA technique were found positive. Similarly, only 50 to 90% of stool specimens seeded with 50,000 oocysts were identified as positive by using the standard FEA technique, compared with a 100% positive rate by the new technique. The new stool concentration procedure provides enhanced detection of Cryptosporidium oocysts in all stool samples.


Baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays.

A recombinant baculovirus that expresses the nucleoprotein gene of measles virus (Edmonston vaccine strain) under the transcriptional control of the polyhedrin promoter was generated. The expressed protein (B-MVN) comigrated with the authentic viral nucleoprotein as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was phosphorylated. The B-MVN protein proved to be reactive with monoclonal antibodies in radioimmunoprecipitations, and it was immunogenic, eliciting in mice antisera that recognized the native nucleoprotein. In addition, the B-MVN protein was evaluated as a replacement source of antigen for whole virus in enzyme immunoassays (EIAs) for detection of measles virus-specific immunoglobulin M (IgM) and IgG antibodies. A capture IgM EIA with the B-MVN protein as antigen detected specific IgM antibodies in 18 (72%) acute- and all convalescent-phase specimens from 25 clinical measles cases and exceeded 99% specificity with 120 control specimens. An indirect IgG EIA with the B-MVN protein detected specific IgG antibodies in 129 of 131 (98%) serum specimens with antibodies to measles virus, and results obtained from testing 268 additional serum specimens were better correlated with measles virus-neutralizing antibodies than those obtained with a commercial EIA.


Susceptibility testing of Cryptococcus neoformans: a microdilution technique.

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.


A novel polymerase chain reaction method for detection of human immunodeficiency virus in dried blood spots on filter paper.

A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology described for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.


Characterization and comparison of Australian human spotted fever group rickettsiae.

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Rickettsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a new rickettsial species.


Evaluation of the autoSCAN-W/A rapid system for identification and susceptibility testing of gram-negative fermentative bacilli.

The autoSCAN-Walk-Away (W/A) system for identification and susceptibility testing was evaluated for 400 gram-negative fermentative bacteria by using the API 20E (366 isolates) and/or tube biochemical tests as the reference identification system and a frozen microdilution MIC tray system for susceptibility testing. The W/A system performed well for identification of this group of organisms representing 14 genera and 30 species, showing a sensitivity of 96% and results available in 2 h. Of the 16 misidentifications, 6 were with Serratia liquefaciens. A total of 63 isolates (17%) required further tests to complete the identification, compared with 106 (29%) of the isolates which required additional tests for the API 20E identification. Approximately half (32) of the additional tests with the W/A system were required in order to separate Citrobacter diversus from C. amalonaticus. For susceptibility determinations, the W/A system demonstrated an overall agreement of 93% (4,102 determinations) with 40 major errors (0.98%). However, of the 906 resistant organism-drug combinations in the study, there were 115 very major errors, for a false-susceptibility rate of 12.7% of the resistance determinations. Among these very major errors, 80% occurred with piperacillin and the cephalosporins. The W/A system completed the MIC determinations in 7 h; however, the difficulty in detecting resistance with some antimicrobial agents limited the advantages of the rapid susceptibility testing.


Establishment of a particle-counting method for purified elementary bodies of chlamydiae and evaluation of sensitivities of the IDEIA Chlamydia kit and DNA probe by using the purified elementary bodies.

To evaluate the sensitivity of commercially available test kits for detection of chlamydiae, we established a method of purifying Chlamydia trachomatis and Chlamydia pneumoniae elementary bodies (EBs). We then subjected the purified EBs, together with the purified EBs of Chlamydia psittaci, to the IDEIA Chlamydia (IDEIA) and DNA probe test kits to determine the EB numbers at the detection limits. The sensitivities of the test kits were thus compared. The results can be summarized as follows. (i) Intact EBs in the purified preparations were present at 100, 96.3, and 97% for the C. psittaci Cal 10, C. trachomatis L2/434/Bu (L2), and C. pneumoniae TW-183 strains, respectively. The preparations of the L2 and TW-183 EBs contained a few EB envelopes, which reacted with antilipopolysaccharide monoclonal antibodies, as did the intact EBs, indicating that elimination of EB envelopes is not required for testing of the IDEIA kit's sensitivity. (ii) We established a method of counting intact EBs and EB envelopes under a scanning electron microscope after sedimentation of EBs on a coverslip by centrifugation. (iii) The EB numbers per assay at the cutoff level, which is set up in the IDEIA kit, were 9.6 x 10(2), 6.5 x 10(3), and 2.5 x 10(4) for the L2, TW-183, and Cal 10 strains, respectively. When the same EB preparations were applied to the DNA probe kit, the EB number at the cutoff level was 7.5 x 10(3) per assay for the L2 strain, but no reaction occurred for the Cal 10 and TW-183 strains at any EB number, indicating that the DNA probe kit is highly specific for C. trachomatis. Although the IDEIA kit designed for detection of C. trachomatis showed a sensitivity superior to that of the DNA probe, the chlamydial species was not determined by the IDEIA kit.


Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction.

Oligonucleotide probes which match consensus sequences of the repetitive extragenic palindromic (REP) element hybridize to genomic DNA of diverse bacterial species. Primers based on the REP sequence generate complex band patterns with genomic DNA in the polymerase chain reaction (PCR), a technique named REP-PCR. We used REP-PCR with genomic DNA to fingerprint 47 isolates of Citrobacter diversus. Previously, 37 were assigned electrophoretic types (ETs) by multilocus enzyme electrophoresis and 35 were evaluated by using outer membrane protein profiles. Fingerprints were compared by visual inspection and by similarity coefficients (SimCs) based on the number of common bands versus total bands between two given isolates. DNA fingerprints were highly similar visually for patient pairs and outbreak-related sets. SimCs for these were > or = 0.952. Fingerprints of isolates with different ETs generally were distinctive. Among 21 unrelated isolates representing 15 ETs, only 6 of 210 comparisons had SimCs of > or = 0.952. REP-PCR rapidly generated DNA fingerprints which were highly similar for epidemiologically linked isolates of C. diversus and distinct for previously characterized strains within this species. The ability of this method to discriminate between C. diversus isolates with the same biotype was similar to that of multilocus enzyme electrophoresis and outer membrane protein profiles. REP-PCR may be useful in evaluation of apparent outbreaks of this or other bacterial species which possess these extragenic, repetitive elements.


Comparison of two culture approaches, blind passage and dual observation, for detecting Chlamydia trachomatis in various prevalence populations.

Chlamydia trachomatis diagnosis in our laboratory consisted of dual inoculation of shell vials and detection of inclusions by using fluorescein-conjugated monoclonal antiserum; the second culture vial was conventionally used for blind passage when the first vial was negative. We compared the increase in positivity using blind passage with that of a strategy utilizing observation of two stained monolayers (dual observation) without blind passage, in an effort to reduce the reporting time and labor associated with the conventional approach. A total of 4,329 specimens were obtained from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the sexually transmitted disease clinic (1,766 specimens). These specimens were used to compare the two strategies. Blind passage of 1,269 initially culture-negative specimens from the OB-GYN clinic resulted in an additional 6 positive chlamydial diagnoses. In comparison, a similar number of specimens (1,294) from the OB-GYN clinic collected subsequently to the first group were tested by dual observation. There were five additional positive findings. A similar evaluation of specimens from the sexually transmitted disease clinic was performed. Blind passage of 313 initially culture-negative specimens yielded 3 additional positive diagnoses, whereas dual observation of 1,435 similar specimens resulted in 9 positive diagnoses. On the basis of analysis of 4,332 specimens, sensitivity of dual observation is comparable to that of blind passage; labor, cost, and reporting time of dual observation are reduced in comparison to those of blind passage.


Urinary tract infection due to Rahnella aquatilis in a renal transplant patient.

Rahnella aquatilis is an unusual gram-negative rod belonging to the family Enterobacteriaceae. It inhabits fresh water and is rarely isolated from clinical specimens. We report the case of a urinary tract infection caused by this organism in a renal transplant patient.


DNA probe for strain typing of Cryptococcus neoformans.

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.


Comparative study of antipneumocystis agents in rats by using a Pneumocystis carinii-specific DNA probe to quantitate infection.

A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods.