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U.S. Department of Health and Human Services

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FY 2000 Reuse/Sterilization/Disinfection/Infectious Disease Diagnostics

Key words: preclinical, biocompatibility, materials science, sterility, reuse, clinical use, surveillance, risk analysis, end of product life, infectious diseases, in vitro diagnostics

FDA has traditionally not regulated reprocessing of single-use devices, but recently decided that regulation was necessary to assure the safety and efficacy of reprocessed medical devices. The OST Reuse Program is designed to address the issues of safety and efficacy associated with the reuse of devices that were intended as single-use devices (SUD's). Data and other information obtained in this OST research program have contributed to this policy and will help in the final formulation and implementation of the regulations. The experience gained from this program will allow CDRH to understand the problems and ask the right questions related to SUD reprocessing. SUD's were intended by the original equipment manufacturer (OEM) to be discarded after single use and not to be reused on another patient. SUD's used in interventional cardiology and in gastrointestinal procedures are obtained from Walter Reed Army Hospital and the Washington Hospital Center. Difficulties in cleaning these devices indicated unique problems with access to lumens and interstices that may contain blood and tissue. If the device cannot be cleaned, it cannot be reprocessed safely. Consequently, a potential hazard may exist if the device is reused on another patient. These lumens and interstices are narrow and opaque, and protocols to clean and validate cleaning are being developed. Cleaning and sterilization are safety issues. SUD's were also examined for performance (efficacy) and damage associated with use and cleaning. Because of concerns for laboratory personnel safety, the retrieved devices must be disinfected and cleaned prior to being handled and inspected. Some devices were examined after simulated reuse and reprocessing. A large collection of devices and models of these devices are available for further study. Interventional cardiology devices include many models of percutaneous transluminal coronary angioplasty catheters (PTCA) [balloon catheters], balloon inflators, electrophysiology catheters, cardiac ablators of various kinds, ultrasonic imaging catheters, angiography catheters, guiding catheters, revascularization catheters, and balloon catheters for wedge procedures. Each device has its own specific cleaning needs and various materials are used. The gastrointestinal devices include various models of GI biopsy forceps and snares, devices for retrieving gallstones (balloons, snares, ERCP devices), and various GI catheters.

Protocols for cleaning, sterilizing and evaluating performance of these devices are being developed. These studies provide independent data to support review and regulatory decision-making related to the adequacy of reprocessing of SUDs. This research is designed to uncover problems that the reprocessor may inadvertently run into while reprocessing single-use devices and that CDRH needs to be aware of in regulating the practice of reprocessing single use devices. The use of simple techniques, reagents, and readily available equipment is emphasized so that the results are transferred to the actual reprocessing undertaken in hospitals and third party industry. However, newer technologies are also being incorporated to understand important advancements that must be considered for managing risks in the future.

This program has contributed independent data and information that is being used in compliance actions and in device approval considerations. The output from this research played a major role in formulating the CDRH policy that, effective February 14, 2001, third party reprocessors and hospitals must follow. All third party reprocessors had to submit PMA applications for reprocessing Class III devices by February 14, 2001, and must submit 510K applications for other devices by August 14, 2001. The hospitals have until the August date to submit PMA or 510K applications. The research team is active in reviewing these documents. The research team is also actively participating in developing standards for cleaning methods/validation and issues of sterilization.

CDRH continues to be actively involved with the regulatory evaluation of premarket applications for commercial in vitro diagnostic devices. A relatively recent and important component of this effort is the evaluation of premarket nucleic acid-based kits used to detect and identify infectious microorganisms. OST scientists perform laboratory research projects that involve utilizing equipment and methodologies associated with these devices, and this experience enables them to 1) participate effectively in the CDRH regulatory review of the devices, 2) make informed regulatory decisions concerning the safety and efficacy of the devices, and 3) efficiently standardize the associated methodology.

Cleaning and Performance of Single-Use Devices
Key words: reuse of single use devices, cleaning, simulated soils, preclinical

OST continued to study the effects of reprocessing single-use devices for interventional cardiology and gastrointestinal procedures. Cleaning these devices indicated unique problems with access to lumens and interstices containing blood and tissue. This was of special concern in some of the newly acquired devices with lumens that were difficult to recognize, contaminated with biological material, and not accessible for cleaning. Changes in the devices and the presence of lumens are not trivial in developing cleaning protocols. A tenacious soil to simulate biological debris, and which can be introduced into narrow lumens, was developed to validate cleaning protocols. The effect on device performance and materials properties after simulated reuse and cleaning of such soil is leading to new technologies and issues to be addressed in reprocessing.

Standards for Sterilization and Cleaning Methods
Key words: standards, sterilization, cleaning, disinfection, AAMI

OST is involved in writing, reviewing, and approving various national and international standards. "Sterilization of medical devices-requirements for products labeled ‘Sterile’" was finally approved. OST is involved in writing "Bacterial endotoxin-test methodologies, routine monitoring and alternatives to batch testing." Additionally, a new work proposal "Compendium of cleaning reusable medical devices for reprocessing was submitted to the Association for the Advancement of Medical Instrumentation (AAMI). OST staff are involved in AAMI Sterilization Standards Committee/U.S. Technical Advisory Group (TAG)for ISO/TC 198, Sterilization of health care products as members of various working groups (Executive Board, Co-chair of AAMI Sterilization Standards Working Groups/U.S. Sub-TAGs, Microbiological methods, Sterilization Packaging, Decontamination, Washer-Disinfectors, Cleaning).

Developing a PCR-PNA-ELISA Procedure for Rapid Detection of Antibiotic Resistant Strains of Mycobacterium Tuberculosis (MTB)
Key words: Mycobacterium tuberculosis, in vitro diagnostics, polymerase chain reaction, PCR, peptide nucleic acid, PNA, standard.

The emergence of drug-resistant strains of Mycobacterium tuberculosis (MTB) remains a serious public health problem. New methods for the rapid diagnosis of MTB drug resistant strains are needed. OST is developing a polymerase chain reaction (PCR)-peptide nucleic acid (PNA)-based ELISA as a diagnostic method using point mutations in genes associated with isoniazid and rifampin resistance in MTB. PCR with fluorescein-labeled primers is used to amplify specific regions of the mutant or wild-type cloned mycobacterial genes. Biotinylated PNAs bound to streptavidin-precoated microtiter wells are incubated with the (denatured) PCR-amplified mutant or wild-type gene sequences under conditions that allow hybridization to occur between the labeled PNAs and amplified DNA.

After hybridization, color (405 nm) is developed following addition of an anti-fluorescein conjugate and appropriate substrate. Thus far, OST scientists have established the hybridization temperatures (50-55oC) and other experimental conditions suitable for detecting a number of clinically relevant point mutations in the katG and rpoB genes, using PNA containing 15 bases DNA probes. Hybridization of PCR-amplified MTB DNA sequences that contain these point mutations with mutant-specific complementary PNAs result in ~ 6-10 fold increases in ELISA response compared to hybridization using MTB wild-type-specific PNAs. Conversely, wild-type MTB sequences hybridize more efficiently at 50-55oC with wild-type PNAs than with the mutant-specific PNAs. Using the PCR-PNA-ELISA method with these MTB drug-resistant gene model systems, scientists can identify drug-resistant MTB sequences in less than 24 hrs.

During the course of this work, the OST Project Principal Investigator led the development of an important standards guideline. The guideline (E2048 "Standard Guide for Detection of Nucleic Acids of the Mycobacterium Tuberculosis Complex and Other Pathogenic Mycobacteria by the Polymerase Chain Reaction Technique") was developed through ASTM Committee E-48 on Biotechnology in collaboration with DIN (Deutsches Institut fuer Normung = German Institute for Standardization) Committee E3/E9 on Molecular Biological Detection of Mycobacteria. This guideline was published by ASTM in January 2000.

Isolation of High Molecular Weight DNA from Human Brain Tissue
Key words: DNA, DNA isolation, brain, polymerase chain reaction, PCR

A method was developed for the cold-temperature isolation of high-molecular weight DNA from postmortem human brain tissue. Brain tissue samples were taken from different locations of three brains and frozen at -70oC. Small portions of each sample were immersed in liquid nitrogen and ground to a fine powder with mortar and pestle. The powder was allowed to thaw in buffer and then quickly treated with a protein-digesting enzyme followed by standard phenol-chloroform extraction of DNA. Since the extracted and pulverized brain tissues are kept frozen as long as possible before DNA isolation, there is less chance that DNA degradation can occur. The isolated DNA was analyzed by spectrophotometry and shown by agarose gel electrophoresis to be of high molecular weight. The presence of DNA in the isolated samples was also demonstrated by amplifying a segment of the β-actin gene from an aliquot of the preparation by polymerase chain reaction (PCR) using specific primers.

This procedure was developed during a previous investigation that concerned the use of PCR to determine the distribution of HIV-genomic DNA in brains of deceased AIDS patients at different stages of HIV infection. Developing methodology was a collaboration with investigators from the Institute of Molecular Virology, GSF National Research Center for Environment and Health, Neuherberg, Germany. This brain DNA isolation procedure could be useful for PCR studies that require high-molecular weight brain DNA as starting material. An example of such study is the performance of PCR or other DNA-based molecular in vitro diagnostics procedure to determine the extent of central nervous system infection associated with epidemics caused by DNA-containing microorganisms.

The development of a guideline ("Standard Guide for Detection of Nucleic Acid Sequences of the Human Immunodeficiency Virus HIV-1 by the Polymerase Chain Reaction Technique") was associated with this project. This project was led by the OST Project Principal Investigator through ASTM Committee E-48 on Biotechnology in collaboration with DIN (Deutsches Institut fuer Normung = German Institute for Standardization) Committee E9 on Serodiagnosis of Infectious Diseases and Diseases of the Immune System. The guideline is currently up for vote within ASTM for approval.