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Prediction rule for etiology of vague abdominal pain in the emergency room: utility for imaging triage.

RATIONALE AND OBJECTIVES: To determine the predictive value of clinical parameters in patients with nonspecific abdominal pain undergoing computed tomography (CT) evaluation of the abdomen and pelvis in the emergency room (ER). MATERIALS AND METHODS: A cross-sectional study of a total of 164 sequential abdominal CT exams of the abdomen and pelvis during a 4 month period for nonspecific abdominal pain in the ER setting identified 100 abnormal scans in 164 patients (61 men, 103 women) of average age 46 years (range 4-97). Patient demographic characteristics (age, sex, temperature, white blood cell (WBC) count, and presence of peritoneal signs) were recorded at the time of CT examination. Results of the CT studies were correlated with the clinical data and discharge diagnosis to assess their positive predictive value using ordinal logistic regression. RESULTS: There were 17 cases of appendicitis, 9 cases of diverticulitis, 3 neoplasms, 3 abdominal abscesses, 2 pancreatitis, 2 duodenitis, 5 with fluid collections, 1 buttocks abscess, and 1 epiploic appendagitis were diagnosed with CT, 57 patients had unrelated findings on CT (common but not usually associated with vague pain). A diagnosis of appendicitis correlated with; elevated WBC count (>11.5) ( = 0.002), male sex ( = 0.001), and younger age (<25 years old) ( = 0.002). A positive CT correlated with an elevated WBC >11.5 (OR, 7.7; 95% CI, 3.3-18). The presence of peritoneal signs and fever did not correlate with a positive CT finding and diverticulitis had no predictive variables. Alternative diagnoses were correlated with female sex ( = 0.014). The combination of; age, sex, and WBC count allowed for a prediction rule with Area under the receiver operator curve of 0.92 to be generated. CONCLUSION: An elevated white blood cell (WBC) count is strong evidence of the presence of an inflammatory process. Alternative diagnostic considerations should be entertained in the context of a normal WBC count without strong clinical suspicion, particularly in women. The use of these factors alone allowed the construction of a prediction rule that can be used for CT protocol optimization.

Abdominal Pain↗

3D virtual endoscopy of the upper airway: optimization of the scan parameters in a cadaver phantom and clinical assessment.

Our goal was to evaluate the influence of the scan parameters on the 3D virtual endoscopy of the larynx and trachea and the clinical assessment. Helical CT (HiSpeed Advantage; GE, Milwaukee, WI, U.S.A.) of a cadaver phantom was performed with increased collimation (1-10 mm) and pitch (0.5-3). Seventy-two patients with complaints of the upper airways were investigated by virtual endoscopy and their results were compared with the findings of regular endoscopy. Best correlation between virtual endoscopy and anatomical findings, diagnostic quality of the axial slices, and useful longitudinal coverage of the examination were obtained with a collimation of 3 mm and a pitch of 1.5. Space-occupying tumors and stenosis were detected correctly, but the mucous membrane could not be visualized. 3D virtual endoscopy proved to be a valuable method for displaying anatomical structures. For an optimal protocol, a collimation of 3 mm with a pitch of 1.5 is recommended.

Endoscopy↗

Therapeutic optimization including inhaled nitric oxide in adult respiratory distress syndrome in a polyvalent intensive care unit.

OBJECTIVE: To investigate the effects of inhaled nitric oxide (NO) in adult respiratory distress syndrome (ARDS) associated with a therapeutic optimization strategy on oxygen parameters, barotrauma, and evolution in a medical and surgical intensive care unit. DESIGN: Prospective study. MATERIALS AND METHODS: Twenty consecutive patients with ARDS were studied (Murray score 3.6 +/- 0.2). Eleven were surgical patients and nine were medical patients. All fulfilled the extracorporeal membrane oxygenation entry criteria. The APACHE II score predicted mortality was 39%. All were ventilated with FiO2 1 with positive end-expiratory pressure (PEEP) of 11 +/- 1 cm H2O. Therapeutic optimization included permissive hypercapnia, tracheal gas insufflation, prone position, continuous hemofiltration, treatment of infection, and pleural drainage. We used NO continuously inhaled at a concentration ranging from 5 to 10 ppm. MEASUREMENTS AND MAIN RESULTS: After 1 hour, inhaled NO improved PaO2 in all patients except one (78 +/- 11 to 130 +/- 25 mm Hg) (p < 0.05), allowing a reduction of FiO2 and PEEP. After 24 hours, mean pulmonary arterial pressure decreased from 31 +/- 3 to 25 +/- 2 mm Hg (p < 0.05). Systemic hemodynamics were unaffected. Oxygen delivery increased from 531 +/- 135 to 603 +/- 125 mL/minute/m-2 (p < 0.05). Barotraumatic lesions were present in only one patient. Reversal of ARDS was obtained in 16 patients, of whom 14 (70%) were discharged. CONCLUSIONS: This study was shorter to demonstrate an improvement in the survival rate. Nevertheless, these preliminary results are encouraging. Because of its safety, effectiveness, and easy use, inhaled NO should be used as a part of a therapeutic optimization protocol before considering more invasive and expensive procedures, such as extracorporeal respiratory support or intravascular oxygenation.

APACHE↗

Computed tomographic scans of minimally displaced type II odontoid fractures.

OBJECTIVE: To determine the computed tomography (CT) appearance of minimally displaced type II odontoid fractures and the optimal protocols to evaluate these fractures by CT. MATERIALS AND METHODS: The CT scans of five patients with minimally displaced type II odontoid fractures and 71 patients without odontoid pathology were reviewed for signs of fracture. A phantom consisting of a cadaver specimen with a type II odontoid fracture was evaluated with several protocols on four CT scanners. The protocols differed in slice thickness and reconstruction algorithm. Helical scanning was also performed, and parasagittal and coronal reformations were created from each image set. MAIN RESULTS: Multiple cortical disruptions longer than 1 mm were demonstrated on the scans of all five patients with type II odontoid fractures. Only three of the 71 patients without odontoid fractures had multiple cortical disruptions, and none were longer than 1 mm. In the phantom study, thinner sections demonstrated cortical disruptions better than thicker sections. Similarly, images reconstructed with a bone reconstruction algorithm demonstrated the cortical disruptions better than images reconstructed with a soft-tissue reconstruction algorithm. Helical scans (1 mm thick) adequately demonstrated the reduced Type II odontoid fracture. Parasagittal and coronal reformations failed to demonstrate a fracture line through the base of the odontoid process on all image sets. CONCLUSIONS: Cortical disruptions greater than 1 mm and multiple cortical disruptions may be the only findings of odontoid fractures and should suggest the diagnosis. Evaluation of potential type II odontoid fractures is improved as CT section thickness is reduced (down to 1 mm) and is also improved by use of a bone reconstruction algorithm. The apparent absence of a fracture line through the base of the odontoid process on parasagittal or coronal reformations does not rule out the diagnosis.

Cadaver↗

Rapid preparation of 123I-labelled methylene blue and toluidine blue: potential new agents for parathyroid scintigraphy.

Methylene blue and toluidine blue are phenothiazinium dyes used to localize parathyroid glands visually during surgery. For the purpose of assessing the potential of radiolabelled analogues for scintigraphic localization of parathyroids, a rapid and convenient method has been developed for covalently labelling the dyes with iodine-123. Treatment of aqueous methylene blue or toluidine blue with 123I-sodium iodide in the presence of potassium iodate and hydrochloric acid at 100 degrees C for 1 h results in incorporation of 80-90% of the radioiodine into the dye. The chemical and radiochemical impurities are readily removed by passage through a disposable sample preparation column. Using the optimized protocol, a sterile, pyrogen-free multi-dose solution of radioiodinated dye (pH 7, specific activity greater than or equal to 37 MBq mg-1, radioactivity concentration congruent to 37 MBq ml-1, radiochemical purity greater than 99%, overall activity yield available for injection 60-70%) can be prepared within 1.5 h.

Humans↗

A nonviral vector system for efficient gene transfer to corneal endothelial cells via membrane integrins.

BACKGROUND: Genetic manipulation of allografts to suppress their ability to induce rejection is a promising approach for controlling rejection responses. A key to this approach is the development of appropriate DNA vectors. We are developing nonviral DNA vector systems based on synthetic peptides containing an integrin-binding segment for cellular targeting and a polylysine segment for DNA binding. METHODS: Two such peptides have been tested for their ability to deliver the beta-galactosidase reporter gene to the corneal endothelial cells of the rabbit, pig, and man. One peptide was derived from a phage display library, the other from the integrin-binding moiety of the toxin from the American pit viper, Crotalus molossus molossus. Corneas were cultured overnight and then exposed to the DNA/peptide vector under a variety of conditions involving different DNA concentrations, chloroquine concentrations, times of exposure, presence of serum, and presence of polyanion buffers. Expression of the beta-galactosidase gene was determined after 3 additional days in culture. Effects of the treatment on the viability of the endothelium were examined by confocal microscopy. RESULTS: We report that approximately 30% of corneal endothelial cells can be transfected with our optimal protocol using the molossin-based vector. Transfection is dependent on the presence of chloroquine and is inhibited by polyanion buffers such as HEPES. Viability of the corneal endothelium was excellent, except if corneas were incubated at high concentrations of chloroquine (0.5 mM) for prolonged periods (24 hr). CONCLUSIONS: Synthetic peptides containing both an integrin targeting and a DNA-binding moiety are promising as simple and highly versatile DNA vectors for use in corneal transplantation.

Animals↗

Centrilobular necrosis in children after combined liver and small bowel transplantation.

BACKGROUND: Centrilobular necrosis is not an uncommon finding after isolated liver transplantation. In this study, we sought to describe hepatic centrilobular necrosis in children after combined liver and small bowel transplantation (LSBT), and to assess the predictive factors, possible causes, and prognosis. METHODS: Six children aged 4 to 11 years, in whom liver biopsy showed centrilobular necrosis at least once, 3 weeks to 2 years after LSBT, were compared with nine children without this pathology. All six children experienced an acute complication in the few weeks preceding the finding of centrilobular necrosis. In addition, one child had an early arterial thrombosis and one, severe colitis 3 years after LSBT. RESULTS: Centrilobular necrosis was associated with centrilobular swelling, dropout, endotheliitis, and inflammation. Fibrosis developed early and worsened on follow-up biopsy in three children. Portal symptoms of acute rejection were not constant, and there was no ductopenia. Biologic abnormalities were responsive to increased immunosuppression, including mycophenolate in four cases. However, follow-up biopsies showed persistent lesions in five patients, mildly inflammatory in four. Baseline immunosuppression had to be maintained at high levels. No viral infections, vascular compromise (except in one), and autoimmunity were found. We compared the two groups of children for initial diagnosis, age at transplantation, time receiving parenteral nutrition, ischemic time, presence of an associated transplanted colon, number of reoperations and infections, intestinal rejection, and immunosuppression, and found no differences. CONCLUSIONS: This severe manifestation of chronic liver rejection occurred despite the heavy immunosuppression needed for LSBT. The previous acute clinical event could have triggered rejection by modifying the effective immunosuppression at the tissue level. Despite high baseline immunosuppression, histologic lesions persisted and significant fibrosis developed in half the children. We speculate that the lack of induction of tolerance in this particular setting of LSBT could be responsible for constant immune stimulation, thus chronic rejection. The optimal protocol of immunosuppression has yet to be defined to avoid this complication.

Child↗

Uronephrologic nuclear medicine.

Nuclear medicine offers a rapid noninvasive means of evaluating suspected urinary tract disorders. Over the years, radionuclide scintigraphy has gained an important role in the evaluation of the renal transplant, possible obstruction, infection, and renovascular hypertension. During the past year, the importance of nuclear medicine within these areas has been confirmed. The diagnostic value of both diuresis and angiotensin-converting enzyme inhibitor renography is now generally accepted, whereas there is still disagreement about the optimal protocol. Renal scintigraphy is useful in patients with renal infection. Nuclear medical examinations have assumed a dominant role in the control and monitoring of renal transplants owing to their ability to quantify perfusion and function, but they do not obviate graft biopsy.

Humans↗

The current status of intravesical therapy for superficial bladder cancer.

PURPOSE OF REVIEW: To analyse recent advances in intravesical instillation therapy for superficial bladder cancer. RECENT FINDINGS: Although intravesical bacillus Calmette-Guérin has been used for many years in the treatment of superficial bladder cancer, its mechanism of action remains unclear, its poor tolerance remains a problem, the prediction of its efficacy has still to be validated, and its long-term effects on progression and survival are controversial. The exact timing and place of intravesical chemotherapy needs to be better defined, as well as the place of some new molecules. Finally, new approaches need to be explored for overcoming the limitations of the usual intravesical agents. SUMMARY: No dramatic advances have been made in understanding the mechanisms of action of bacillus Calmette-Guérin during the past year. However, a careful dissection of this complex immunological pathway continues and immunological criteria are promising for predicting the response to bacillus Calmette-Guérin. Evidence has been accumulating to suggest that a dose reduction during the initial treatment remains effective and reduces side-effects. In addition, bacillus Calmette-Guérin maintenance therapy is useful for high-risk patients. However, long-term tolerance remains an important issue, and the optimal protocol has not yet been defined. On the other hand, it has been proved that intravesical chemotherapy, when administered early after transurethral resection, is effective in preventing frequent recurrences, whereas maintenance chemotherapy is ineffective. Finally, new approaches, including instillations of activated immune cells or targeted gene therapy, are being explored.

Adjuvants, Immunologic↗

Effects of hormone therapy on brain morphology of healthy postmenopausal women: a Voxel-based morphometry study.

OBJECTIVE: Estrogens are known to be protective in age-associated cognitive changes in humans and in neurodegeneration in animal models. The aim of this study was to evaluate the potential effects of estrogen therapy (ET) on human gray matter volume in vivo. DESIGN: Forty healthy postmenopausal women underwent three-dimensional high-resolution magnetic resonance imaging: 17 were never treated, 16 were currently receiving ET, and 7 had had ET in the past. Voxel-based morphometry (VBM) with SPM2 was used, according to an optimized protocol, to compare women under past and current ET to those never treated. Significance threshold was set at P = 0.01, corrected by false discovery rate. RESULTS: Voxel-based morphometry indicated that estrogen use was associated with greater gray matter volumes in the whole group of treated women, which included the cerebellum (cluster size, Z coordinates: 5,527; 5.15; -14 -54 -10), the amygdaloid-hippocampal complex (left: 19; 3.55; -22 -4 -18; right: 45; 3.61; 16 -6 -16), and extended to the frontal, temporal, parietal, and occipital neocortex. The comparison current ET versus past ET use showed that women who underwent treatment in the past had greater volumes of gray matter compared to women under current treatment. CONCLUSIONS: ET might slow down age-related gray matter loss in postmenopausal women. The structures that exhibited greater volume in association with ET included the cerebellar and cerebral cortices and, typically involved in Alzheimer's disease, the medial temporal structures and the temporoparietal junction.

Aging↗

Cervical spine clearance in unconscious traumatic brain injury patients: dynamic flexion-extension fluoroscopy versus computed tomography with three-dimensional reconstruction.

BACKGROUND: An optimal protocol for clearing the cervical spine in unconscious patients with traumatic brain injury remains controversial. Protocols include plain radiographs and computed tomography (CT), and ligamentous injury may be identified with flexion-extension radiographs. We questioned whether cervical CT with three-dimensional (3D) reconstructions may obviate the need for flexion-extension radiology in the detection of occult ligamentous injury. METHODS: Between July 1999 and November 2001, 276 unconscious traumatic brain injured patients admitted to The Alfred Hospital received cervical spine plain radiographs, CT with 3D reconstructions, and dynamic flexion-extension X-ray studies with fluoroscopy as part of a routine protocol. These patients were identified from a prospective intensive care unit database and all radiology reports were reviewed. RESULTS: Dynamic flexion-extension X-ray studies with fluoroscopy identified no new fractures or instability; there were no instances of true-positive results. Dynamic flexion-extension was true-negative in 260 of 276 (94%) patients, falsely positive in six patients (2.2%) and falsely negative in one (0.4%) patient. In nine patients, dynamic flexion-extension was inadequate. CONCLUSION: Dynamic flexion-extension X-ray studies with fluoroscopy delayed cervical spine clearance and were almost always reported as normal. In a cervical spine clearance protocol for unconscious traumatic brain injury patients, dynamic flexion-extension X-ray studies with fluoroscopy did not identify any patients with cervical fracture or instability not already identified by plain radiographs and fine-cut CT (C0 to T2) with 3D reconstructions.

Algorithms↗

System performance of multislice spiral computed tomography.

Multislice spiral CT offers many new possibilities for clinical CT imaging. Drastically increased scan speeds and z-resolution, respectively, as well as applications such as cardiac CT that have become feasible for the first time in routine clinical use. The concept of multiple simultaneously acquired slices yields image quality equivalent or better than single-slice spiral CT. Especially, there is no dependence on spiral pitch, neither with regard to noise nor to slice sensitivity. The reconstructed slice width can be chosen freely and retrospectively, which offers additional flexibility when evaluating optimal protocols for various kinds of examinations. Three-dimensional isotropic resolution can be achieved routinely with examinations fast enough to scan in a single breath hold (Fig. 9). Without any drawbacks in image quality, MSCT in combination with online tube current modulation can reduce patient dose. In some body regions, dose is decreased to 50% compared to a scan with constant tube current. One of the most promising new applications is the dedicated ECG-gated cardiac interpolation 180 degrees MCI, which allows four-dimensional (4-D) imaging of the heart. The complete beating heart can be reconstructed in well-defined phases of the heart cycle, thereby adding high temporal resolution to isotropic 3-D spatial resolution (for more examples refer to http://www.imp.uni-erlangen.de/e/research/cardio/).

Algorithms↗

Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa.

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.

Animals↗

High level cell-free expression and specific labeling of integral membrane proteins.

We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein 'remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced by formation of the proposed alpha-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins.

Base Sequence↗

Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR.

A real-time PCR assay for Clostridium difficile was developed, based on the tcdB gene, which detected all known toxinogenic reference strains (n = 45), within 30 serogroups and 24 toxinotypes. The analytical sensitivity was 1 x 10(3) CFU/mL, and the detection limit in faeces was 1 x 10(5) CFU/g. The optimal protocol for DNA extraction from faecal samples involved use of the MagnaPure system with a Stool Transport and Recovery (STAR) buffer pre-treatment. In a 1-month prospective study of 85 patients with diarrhoea, the sensitivity, specificity and positive and negative predictive values of the assay were 100%, 94%, 55% and 100%, respectively, compared with the standard cell cytotoxicity assay.

Adult↗

The failed renal transplant: in or out?

With the increased number of renal transplants being performed in the United States, there are also increasing numbers of early and long-term allograft failures. Patients with failed allografts require reentry into the end-stage renal disease (ESRD) system. If an allograft is producing little in the way of morbidity, it seems reasonable to continue the patient on low-dose immunosuppressive therapy to avoid sensitization while leaving the allograft in. This is particularly true if the allograft is providing enough renal function to keep the patient off dialysis. However, if the patient is having serious morbidity that may be due to the retained, failed kidney transplant, it may be necessary to remove the allograft kidney, which often shows ongoing inflammation. This may restore erythropoietin responsiveness and contribute to the overall benefit of the patient. The optimal protocol for withdrawal of immunosuppression in this circumstance has not been established by any scientific studies, and the effect of allograft nephrectomy on sensitization status and the ability to receive a crossmatch-negative retransplant are controversial. The author's personal approach is presented.

Graft Rejection↗

Estimating blood needs for very-low-birth-weight infants.

BACKGROUND: Red blood cell (RBC) transfusions are crucial for the care of very-low-birth-weight (VLBW) infants. These infants frequently require multiple, small-volume RBC transfusions, with potential exposure to multiple donors. Optimal protocols provide dedicated RBC units to reduce exposures and avoid RBC wastage. STUDY DESIGN AND METHODS: This study was a retrospective, single-institution review of RBC transfusions in VLBW infants. The RBC volume transfused during the entire hospitalization (VTH) and that transfused at 35 days were determined for all infants, 401 to 1250 g at birth, admitted to a Level III neonatal intensive care unit from January 1, 2000, through December 31, 2002. Multivariable models identified perinatal factors associated with volume transfused. RESULTS: The 640-infant cohort had a median birth weight (BW) of 818 g and gestational age (GA) of 26 weeks. Most infants (546 or 85%) required at least one RBC transfusion. Median number of RBC transfusions was 3 (range, 0-30) and median volume transfused was 82 mL (range, 9-737 mL). Of 328 infants who received all transfusions within a 35-day period, only 5 (1.5%) required at least 200 mL. VTH was inversely related to BW and GA. Multivariable models identified BW, GA, age at first transfusion, and use of inotropes as variables associated with higher volume transfused. CONCLUSION: Few VLBW infants use an entire RBC unit. One dedicated unit shared by two or more infants should meet their transfusion needs. GA, BW, and markers of illness severity predict increased RBC volume requirements.

Body Weight↗

Leishmania mexicana amazonensis: differential display analysis and cloning of mRNAs from attenuated and infective forms.

The virulence of Leishmania mexicana is determined by the concerted action of several parasite molecules. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth media. Both virulent and attenuated variants of the parasite cells were cloned. The differential display reverse transcription-polymerase chain reaction technique was employed to understand whether this natural attenuation of the parasite cells is accompanied by differential expression of selected genes in those cells. Twelve different dinucleotide-anchored oligo(dT) antisense primers were used to make cDNAs from poly(A)+ mRNAs isolated from a clonal population of virulent and avirulent cells following a protocol optimized for Leishmania mRNAs. Those cDNAs were subjected to amplifications using each of the three different arbitrary decanucleotide primers and the corresponding anchored oligo(dT) primer. This procedure revealed four virulent-specific cDNA probes and one avirulent-specific cDNA probe. Differential expressions of these genes were confirmed by northern hybridization using the cloned cDNA probes. These results indicate that differential expression of genes may be the key in determining the molecular basis of leishmanial virulence.

Animals↗