Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces

Background

HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV – DNA by Real time – PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals.

Methods

Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector

Results

Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy.

Conclusion

HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time – PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.Key Words: Real time – PCR, Chronic Hepatitis B, HBV – DNA, Antivirals     

Relationship between lipophilicity of BCS class III and IV drugs and the functional activity of peroral absorption enhancers

Absorption enhancers (AEs) have been shown to be specific in permeation enhancement capabilities because of which they increase absorption of some drug molecules more than others. Present study was designed to investigate the relationship between lipophilicity of drug molecules and the absorption enhancement potential of AEs. Four drug molecules of different lipophilicity were selected as model compounds, namely, cefotaxime sodium, ceftazidime pentahydrate, lovastatin and cyclosporin A. Their apparent permeability coefficients in the absence and presence of three classes of AEs (fatty acids, cyclodextrins, and bile salts) were determined using in vitro everted rat intestinal sac absorption model. Significant relationship was observed between log P of drug and absorption enhancement ratios by AEs. This relationship was found to be functionally directly or indirectly proportional depending upon nature of AE and explain the mechanism of permeation enhancement.     

Cervical spine assessment in the unconscious trauma patient: a major trauma service's experience with passive flexion-extension radiography

BACKGROUND: There is no consensus on the most appropriate method of cervical spine assessment in unconscious trauma patients. Passive flexion-extension imaging is one option for further investigating unconscious patients whose plain cervical radiographs are normal. This study examines the usefulness of this passive imaging in investigating for occult cervical injury. METHODS: All unconscious patients admitted to The Alfred Trauma Intensive Care Unit over 1 year (January 1-December 31, 1998), who could not be clinically assessed within 48 hours in regard to their cervical spine, were identified. Results of passive flexion-extension radiography were compared with final injury status and clinical outcome as determined by retrospective review of the imaging reports, radiographic films, and case notes. RESULTS: One hundred twenty-three patients with normal three-view plain radiographs proceeded to passive functional investigation. These were false-negative in four of the seven patients with cervical spine injuries at presentation. No patients suffered any adverse neurologic events from their delayed diagnoses or from the flexion-extension procedure. CONCLUSION: Passive flexion-extension imaging has inadequate sensitivity for detecting occult cervical spine injuries. Although no patients suffered adverse neurologic complications, the potential for devastating consequences from missed cervical injury has resulted in the removal of passive flexion-extension imaging from the screening protocol.     

Emergency surgery after percutaneous transmitral commissurotomy: operative versus echocardiographic findings, mechanisms of complications, and outcomes

OBJECTIVE: This study was undertaken to determine the clinical profile of patients undergoing emergency surgery after balloon mitral valvotomy, to note operative findings and compare them with those of transthoracic echocardiography, to describe the mechanisms of complications, and to describe outcomes. METHODS: A retrospective study was undertaken of patients requiring emergency surgery after percutaneous mitral valvotomy with an Inoue balloon from January 1990 to December 2003. The data analyzed included demographic variables, causes and clinical presentations of complications, and outcome. In 14 consecutive cases of mitral regurgitation, an observational study comparing the operative findings with echocardiography was also undertaken. RESULTS: In 1388 cases of valvotomy, complications necessitating urgent surgery occurred in 31 cases (2.2%). Acute mitral regurgitation occurred in 23 cases (74.2%), and cardiac tamponade occurred in 8 cases (25.8%). Mitral regurgitation was due to leaflet tearing in all cases: anterior leaflet in 20 cases and posterior leaflet in 3 cases. Hypotension, orthopnea, and pulmonary edema were the clinical presentation for mitral regurgitation. Transthoracic echocardiography underestimated the severity of mitral valve pathology. Bilateral severe commissural fusion and pliable leaflet with paracommissural calcium was seen in anterior leaflet tearing. Cardiac tamponade with hemodynamic compromise occurred as a result of left atrial perforation in 6 cases, right atrial perforation in 1 case, and left ventricular perforation in 1 case. High septal puncture led to atrial perforation. Operative mortality was 9.6%, and low cardiac output developed in 29%. CONCLUSION: Acute mitral regurgitation and cardiac tamponade were the causes of emergency surgery after balloon valvotomy. Transthoracic echocardiography underestimated the severity of valve pathology.     

Arachnoiditis associated with arachnoid cyst formation and cord tethering following myelography: Magnetic resonance features

The reported incidence of clinically significant arachnoiditis following oil‐based contrast media myelography is approximately 1%. The incidence of arachnoiditis in the dorsal spine is even more rare. Acquired arachnoid cyst formation can occur with arachnoiditis of various aetiologies. A case is presented of a patient with chronic backache, radiculopathy, weakness and muscle wasting, who had undergone myelography with oil‐based contrast media about 28 years ago. There was no history of spinal surgery, spinal canal stenosis, disc disease or trauma, all of which are known to increase the risk of arachnoiditis. Magnetic resonance imaging features are presented in this case of arachnoiditis with arachnoid cyst formation, and cord tethering.     

Graded contribution of retinal maturation to the development of oxygen-induced retinopathy in rats

PURPOSE: Newborn rats exposed to hyperoxia during the first days of life have been shown to exhibit not only vasculopathy but also permanent changes in the structure and function of the retina. Given that the rat retina is immature at birth and that the maturation process continues until the opening of the eyes at 14 days of life, this study was conducted to investigate the susceptibility of the retina to oxygen toxicity as a function of the degree of retinal maturity reached at the time of oxygen exposure. METHODS: Newborn rats were exposed to hyperoxia during selected postnatal day intervals. Scotopic electroretinograms were recorded at 30 and 60 days of age, and retinal histology was obtained at the end of the study. RESULTS: There was a strong correlation between the duration of the hyperoxic event and the structural and functional consequences in the retina. However, the repercussions were significantly more profound when the exposure to oxygen occurred within the second week of life (6-14 days), compared with earlier (0-6 days) or later periods (14-28 days). CONCLUSIONS: The results strongly suggest that the structural and functional retinal changes secondary to postnatal hyperoxia are not only the direct consequence of exposure to high levels of oxygen (i.e., free radicals), but also are determined by the level of retinal maturity reached at the time of oxygen exposure. The results also indicate that the structural anomalies precede the functional impairments.     

Nuclear type-ii [h-3] estradiol binding-sites in mcf-7 human breast-cancer cells - binding interactions with 2,6-bis-([3,4-dihydroxyphenyl]-methylene)cyclohexanone esters and inhibition of cell-proliferation

Previous studies from this laboratory demonstrated that 2,6-bis-([3,4-dihydroxyphenyl]methylene)cyclohexanone (BDHPC) and related compounds mimic methyl p-hydroxyphenyllactate (MeHPLA) as endogenous ligands for nuclear type II [H-3]estradiol binding sites. Occupancy of type II sites by these agents results in the inhibition of malignant cell proliferation in vitro and mammary tumor growth in vivo. The present studies were designed to assess the effects of BDHPC esterification on type II site binding interactions in uterine nuclei and in cultured MCF-7 human breast cancer cells in vitro. The results of these experiments demonstrate that in rat uterine nuclear fractions BDHPC acetate (Kd approximately 100 nM) interacts with type II sites with a 100-fold lower affinity than BDHPC (Kd approximately 0.9 nM) and BDHPC benzoate failed to inhibit [H-3]estradiol binding under these experimental conditions. Conversely, BDHPC and BDHPC acetate displayed very similar binding affinities for type II sites in cultured MCF-7 human breast cancer cells and there was a direct correlation between nuclear type II site occupancy and the inhibition of cellular proliferation by these two compounds. BDHPC benzoate failed to interact with type II sites or inhibit MCF-7 cell proliferation. Taken together, these results suggested that BDHPC acetate, but not BDHPC benzoate, was being hydrolyzed by esterases in MCF-7 cells, releasing the free parent compound. This conclusion was supported by the observations that incubation of BDHPC acetate in mammary tumor cytosol preparations resulted in essentially quantitative hydrolysis to BDHPC as determined by thin layer chromatography (TLC) and by high performance liquid chromatography (HPLC) analysis of tumor cytosol extracts. Conversely, BDHPC benzoate was not hydrolyzed by tumor esterases which is consistent with the inability of this compound to bind to type II sites or inhibit MCF-7 human breast cancer cell proliferation. These experiments confirm and extend the hypothesis that esterase hydrolysis of MeHPLA related compounds represents an important biological step involved in the control of the biological activity of type II site agonists which appear to regulate malignant cell proliferation through this binding interaction.