Application of lot quality assurance sampling for leprosy elimination monitoring--examination of some critical factors

BACKGROUND: The concept of elimination of an infectious disease is different from eradication and in a way from control as well. In disease elimination programmes the desired reduced level of prevalence is set up as the target to be achieved in a practical time frame. Elimination can be considered in the context of national or regional levels. Prevalence levels depend on occurrence of new cases and thus could remain fluctuating. There are no ready pragmatic methods to monitor the progress of leprosy elimination programmes. We therefore tried to explore newer methods to answer these demands. With the lowering of prevalence of leprosy to the desired level of 1 case per 10000 population at the global level, the programme administrators' concern will be shifted to smaller areas e.g. national and sub-national levels. For monitoring this situation, we earlier observed that lot quality assurance sampling (LQAS), a quality control tool in industry was useful in the initially high endemic areas. However, critical factors such as geographical distribution of cases and adoption of cluster sampling design instead of simple random sampling design deserve attention before LQAS could generally be recommended. The present exercise was aimed at validating applicability of LQAS, and adopting these modifications for monitoring leprosy elimination in Tamil Nadu state, which was highly endemic for leprosy. METHODS: A representative sample of 64000 people drawn from eight districts of Tamil Nadu state, India, with maximum allowable number of 25 cases was considered, using LQAS methodology to test whether leprosy prevalence was at or below 7 per 10000 population. Expected number of cases for each district was obtained assuming Poisson distribution. Goodness of fit for the observed and expected cases (closeness of the expected number of cases to those observed) was tested through chi(2). Enhancing factor (design effect) for sample size was obtained by computing the intraclass correlation. RESULTS: The survey actually covered a population of 62157 individuals, of whom 56469 (90.8%) were examined. Ninety-six cases were detected and this number far exceeded the critical value of 25. The number of cases for each district and the number of cases in the entire surveyed area both followed Poisson distribution. The intraclass correlation coefficients were close to zero and the design effect was observed to be close to one. CONCLUSIONS: Based on the LQAS exercises leprosy prevalence in the state of Tamil Nadu in India was above 7 per 10000. LQAS method using clusters was validated for monitoring leprosy elimination in high endemic areas. Use of cluster sampling makes this method further useful as a rapid assessment procedure. This method needs to be tested for its applicability in moderate and low endemic areas, where the sample size may need increasing. It is further possible to consider LQAS as a monitoring tool for elimination programmes with respect to other disease conditions.     

4-Chloro-benzo[F]isoquinoline (CBIQ) activates CFTR chloride channels and KCNN4 potassium channels in Calu-3 human airway epithelial cells

1 Calu-3 cells have been used to investigate the actions of 4-chloro-benzo[F]isoquinoline (CBIQ) on short-circuit current (SCC) in monolayers, whole-cell recording from single cells and by patch clamping. 2 CBIQ caused a sustained, reversible and repeatable increase in SCC in Calu-3 monolayers with an EC50 of 4.0 microm. Simultaneous measurements of SCC and isotopic fluxes of 36Cl- showed that CBIQ caused electrogenic chloride secretion. 3 Apical membrane permeabilisation to allow recording of basolateral membrane conductance in the presence of a K+ gradient suggested that CBIQ activated the intermediate-conductance calcium-sensitive K(+)-channel (KCNN4). Permeabilisation of the basolateral membranes of epithelial monolayers in the presence of a Cl- gradient suggested that CBIQ activated the Cl(-)-channel CFTR in the apical membrane. 4 Whole-cell recording in the absence of ATP/GTP of Calu-3 cells showed that CBIQ generated an inwardly rectifying current sensitive to clotrimazole. In the presence of the nucleotides, a more complex I/V relation was found that was partially sensitive to glibenclamide. The data are consistent with the presence of both KCNN4 and CFTR in Calu-3. 5 Isolated inside-out patches from Calu-3 cells revealed clotrimazole-sensitive channels with a conductance of 12 pS at positive potentials after activation with CBIQ and demonstrating inwardly rectifying properties, consistent with the known properties of KCNN4. Cell-attached patches showed single channel events with a conductance of 7 pS and a linear I/V relation that were further activated by CBIQ by an increase in open state probability, consistent with known properties of CFTR. It is concluded that CBIQ activates CFTR and KCNN4 ion channels in Calu-3 cells.     

Spinal schwannoma with acute subarachnoid hemorrhage: a diagnostic challenge

We report a case of spinal intradural schwannoma presenting with intracranial subarachnoid hemorrhage. Cerebral angiography studies were negative, but MR imaging of the spine revealed a large hemorrhagic tumor in the thoraco-lumbar junction. The tumor was misdiagnosed as ependymoma of the conus medullaris. This case illustrates the importance of a high index of suspicion for spinal disease in angiographically-negative subarachnoid hemorrhage and pitfalls in MR diagnosis of thoraco-lumbar tumors.     

Bioadhesive tablets for controlled transdermal delivery of drugs

Transdermal bioadhesive tablets were formulated by the direct compression method and evaluated. Carboxyfluorescein (CF) was used as a model permeant. The compression parameters were kept consistent and the composition of the formulation was varied to alter the release rate of the drug from the formulations. The dosage form was evaluated for physicochemical, adherence, and in vitro diffusion parameters. The transdermal flux of the drug decreased with the increasing ratio of Ethyl cellulose (EC)/hydroxypropyl cellulose (HPC). The test formulations were subjected to pharmacokinetic studies in mice. The formulations were able to maintain a steady state plasma concentration up to 12 hours. They were found to be safe for transdermal use as interpreted from skin irritation studies carried out on rabbits.     

Drug-drug interaction studies on first-line anti-tuberculosis drugs

The purpose of this study was to carry out drug-drug compatibility studies on pure first line anti-tuberculosis drugs, viz., rifampicin (R), isoniazid (H), pyrazinamide (Z), and ethambutol hydrochloride (E). Various possible binary, ternary, and quaternary combinations of the four drugs were subjected to accelerated stability test conditions of 40 degrees C and 75% relative humidity (RH) for 3 months. For comparison, parallel studies were also conducted on single drugs. Changes were looked for in the samples drawn after 15, 30, 60, and 90 days of storage. Analyses for R, H, and Z were carried out using a validated HPLC method. The E was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as it does not absorb in ultraviolet (UV). All single pure drugs were relatively stable and showed only 3%-5% degradation under accelerated conditions for 3 months. However, significant interactions were observed in case of the drug mixtures. In particular, ternary and quaternary drug combinations containing R and H along with Z and/or E were very unstable, showing 90%-95% and 70%-75% loss of R and H, respectively. In all these cases, isonicotinyl hydrazone (HYD) of 3-formylrifamycin and H was found to be the major degradation product. In case of RE and RZE mixtures, where H was absent, 3-formylrifamycin was instead the key degradation product. Another unidentified peak was observed in the mixture containing RZE. Apart from these chemical changes, considerable physical changes were also observed in pure E and the mixtures containing E, viz., RE, ZE, RHE, RZE, and RHZE. In addition, significant physical changes associated with noteworthy loss of H and E were also observed in mixtures containing HE and HZE. The present study thus amply shows that the four primary anti-tuberculosis drugs, when present together, interact with each other in a multiple and complex manner.     

Rapid and sensitive HPLC method for the estimation of doxorubicin in dog blood--the silver nitrate artifact

A rapid and sensitive high performance liquid chromatographic (HPLC) assay utilizing fluorimetric detection (excitation at 480 nm, emission at 560 nm) for the determination of doxorubicin in dog blood was developed and validated. Treatment of blood samples containing doxorubicin with AgNO3 (as protein precipitant) resulted in appearance of an additional peak in the chromatogram of doxorubicin at 11.5 min along with the parent peak (tR = 5.5 min). The latter peak was not found when treated with other protein precipitants such as trichloroacetic acid and methanol. Construction of a calibration curve based on the area of the first peak alone did not result in linearity of the curve. However, summation of areas of both peaks resulted in a curve with good linearity and coefficient of determination (R2 = 0.9985). Appearance of the second peak may be due to the interaction of doxorubicin with cellular components of blood in the presence of AgNO3 leading to the formation of a complex with reduced polarity. Analysis of the quality control samples showed good accuracy (96.7-100.42) and precision (RSD = 2.6-5.7%). The proposed method could be advantageous in estimation of doxorubicin incorporated into targeted delivery systems that concentrate in blood cells and quantify the absolute blood concentration of doxorubicin.     

Chitosan-based mucoadhesive microspheres of clarithromycin as a delivery system for antibiotic to stomach

The objective of the present study was to develop chitosan-based mucoadhesive microspheres of clarithromycin to provide prolonged contact time for drug delivery of antibiotics to treat stomach ulcers. Microspheres based mucoadhesive formulation were extensively evaluated and characterized for in vitro performance followed by investigation of in vivo pharmacokinetics in rats. Microspheres were prepared by emulsification technique using glutaraldehyde as a crosslinking agent. Formulation conditions were optimized for percent drug entrapment and mucoadhesion, by varying different formulation and process parameters like drug to polymer ratio, concentration of crosslinking agent and time of crosslinking. Prepared microspheres were evaluated extensively for particle size, percent drug entrapment, swelling kinetics, in vitro mucoadhesion using rat stomach membrane and in vitro drug release studies. In vitro permeation studies across rat stomach membrane were carried out to determine diffusion parameters and drug retention in the stomach membrane of the formulation and the plain drug. Finally in vivo performance of microsphere formulation in comparison to plain drug was evaluated by pharmacokinetic studies in albino rats. Drug entrapment upto 74% was obtained. Swelling studies indicated that with an increase in cross-linking, the swelling ability decreased. The in vitro drug release and in vitro mucoadhesion studies showed a dependence on the extent of cross-linking and concentration of chitosan. Extent of cross-linking exhibited an inverse relation to drug release rate as well as mucoadhesion, whereas polymer concentration exhibited an inverse correlation with drug release while linear relationship with mucoadhesion (up to 86%). In vitro permeation studies across stomach tissue showed higher accumulation of drug in the stomach tissue with microspheres formulation as compared to that of free drug. This is evident from higher value of K (partition coefficient) and Qm/Csf values for microspheres (68.34 and 106.42X10(3), respectively) as compared to that of free drug (1.86 and 173.00, respectively). These findings when analyzed showed an increase in the bioavailability of clarithromycin from microsphere formulation as compared to plain drug suspension in vivo, with AUC 0-->alpha being 91.7 (microg h/ml)and 24.9 (microg h/ml) respectively. Results of the study demonstrated good mucoadhesion of the microspheres with the stomach mucosa as well as higher accumulation of drug in the stomach membrane. Microspheres also exhibited sustained release of drug. Thus chitosan microspheres appear, technically, promising mucoadhesive drug delivery systems for delivering clarithromycin to treat stomach ulcers.     

Melanin: dietary mucosal immune modulator from Echinacea and other botanical supplements

The agents responsible for the therapeutic effects of many botanical supplements have not been established in spite of their popularity. Here we show that melanin is a previously unrecognized immunostimulatory compound that is a major component of botanicals traditionally used to enhance immune function. While melanin is present in commonly consumed vegetables, its specific activity is several orders of magnitude less than melanin extracted from these botanicals. The major reason that this agent has eluded detection is its solvent-specific requirement for extraction/solubility. Melanin activates NF-kappa B in monocytes in vitro through a toll-like receptor 2-dependent process. Ingestion of melanin by mice for four days increases production ex vivo of interferon-gamma by spleen cells and IgA and interleukin-6 by Peyer's patch cells. The identification of this new class of mucosal immune stimulants will allow further characterization of botanical products and advances our understanding of the basis for their traditional use.     

Neoadjuvant chemotherapy in the management of sebaceous gland carcinoma of the eyelid with regional lymph node metastasis

A 55-year-old Asian Indian woman who had recurrent sebaceous gland carcinoma of the left lower eyelid with orbital extension and regional lymph node metastasis was treated with neoadjuvant chemotherapy, using a combination of carboplatin and 5-fluorouracil. Eyelid-sparing orbital exenteration was performed after 3 cycles of chemotherapy, followed by radiotherapy to the regional lymph nodes. Subsequently, 3 cycles of adjuvant chemotherapy were administered. Significant eyelid and orbital tumor volume reduction was achieved with neoadjuvant chemotherapy, making eyelid-sparing orbital exenteration possible. Chemotherapy also spared the patient from radical neck dissection. The patient had limited morbidity and was free of local, regional, and systemic disease at 26 months of follow-up.