Lectures in clinical microbiology: Control Procedures in Clinical Microbiology Labora...

Lectures in clinical microbiology: Control Procedures in Clinical Microbiology Labora...: Remedial action to be taken when calibration or control results fail to meet the laboratory's criteria for acceptability. -Limitatio...

Control Procedures in Clinical Microbiology Laboratory

Remedial action to be taken when calibration or control results fail to meet the laboratory's criteria for acceptability.
-Limitations in methodologies, including interfering substances
Reference or normal ranges
-Imminent life-threatening laboratory results or panic (critical values. Must be informed at once by telephone to doctor e.g. Meningococci in direct gram of CSF.
-Pertinent literature references
Appropriate criteria for specimen storage and preservation to ensure specimen integrity until testing is completed.
The laboratory's system for reporting patient results including, when appropriate, the protocol for reporting panic values. Description of the steps to be taken in the event that a test system becomes inoperable. Criteria for the referral of specimens including procedures for specimen submission and handling
Establishment and Verification of Method Performance Specifications
Prior to beginning to report patient results, the laboratory must establish and verify (for all tests and methods) performance specifications, including -----Accuracy Precision/Analytical Sensitivity Analytical Specificity
-If applicable, the laboratory must also verify the reportable range of patient results as well as the reference (normal) range.
-When a new procedure is implemented, the laboratory must demonstrate, prior to reporting patient results, that it can obtain the performance specifications comparable to the manufacturer's established specifications.
-The laboratory must also verify the manufacturer's reference range for the laboratory's population. The laboratory must then establish calibration and quality control procedures based on the verified performance specifications. Each step must be documented in the laboratory's records.
Equipment Maintenance and Function Checks
The laboratory is required to perform equipment maintenance and function checks (electronic, mechanical and operational). These checks are considered to be necessary for proper test performance and result reporting to assure accurate and reliable test results and reports. The requirements for systems approved by the FDA include maintenance performance as specified by the manufacturer and with at least the frequency specified by the manufacturer. All maintenance performed by the laboratory should be documented. The same applies to system function checks - follow the manufacturer's instructions for both the checks and frequency and document each step
Calibration and Calibration Verification
The laboratory is required to substantiate the continued accuracy of each of its test methods, throughout its reportable range for patients, through calibration and calibration verifications
The reportable range of patient test results is the range of testresult values over which the laboratory can establish or verify the accuracy of the instrument, kit or test system measurement response.
Calibration is the process of testing and adjusting an instrument, kit or test system to provide a known relationship between the measurement response and the value of the substance that is being measured by the test procedure
Calibration verification is the assaying of calibration materials in the same manner as patient samples to confirm that the calibration of the instrument, kit or test system has remained stable throughout the laboratory's reportable range for patient test results.
The manufacturer's instructions should be followed, along with using the materials specified by the manufacturer. Each step should be documented
Control Procedures
On a routine basis, the laboratory must perform control procedures to monitor the stability of the methods or systems utilized by the laboratory. Control and calibration materials indirectly assess the accuracy and precision of patient test results. At a minimum, the manufacturer's instructions are to be followed. The laboratory must:
Test quality control samples in the same fashion as patient specimens
Determine the statistical parameters (e.g., mean , standard deviation) for each lot number through repetitive testing. The stated values of an assayed control material may be used as the target values provided the stated values correspond to the methodology and instrumentation employed by the laboratory and are verified by the laboratory
Accept control results only when the laboratory's criteria for acceptability is met.
• Conduct reagent and supply checks for each shipment or batch of reagents, discs, stains, antiserum and identification system when opened. The checks should include positive and negative reactivity, as well as graded reactivity, if applicable. The laboratory must test staining material each day of use to ensure predicted staining characteristics
• For microbiology media, the laboratory must check for sterility, ability to support growth and, as applicable, selectivity/inhibition and/or biochemical response. The laboratory may use the manufacturer's control checks provided the manufacturer's checks meet the National Committee for Clinical Laboratory Standards (NCCLS) for media quality control.
• Document the physical characteristics of the media to confirm that the media has not been compromised. Report deterioration to the manufacturer.
• Follow the manufacturer's specifications for using the media.
Remedial Actions
• The laboratory must establish policies and procedures for remedial actions for quality control failures and apply them as needed to maintain accurate and reliable patient test results and reports. The laboratory must document when
• Test systems do not meet the established performance specifications. Examples include when equipment or methodologies perform outside established parameters and when patient results are outside of the reportable range
• Control or calibration results fail to meet established criteria. When this occurs, patient results tested between the previous acceptable and the current unacceptable run must be evaluated to determine if the patient results had been affected. The laboratory must take remedial action to ensure the reporting of accurate and reliable results. The laboratory cannot report patient test results within its regular time frames. The laboratory must determine (based on the urgency of the tests requested) the need to notify the appropriate individual of the delay.
• Errors are detected in reported patient test results.
o The laboratory must promptly notify the authorized person ordering the test or the individual utilizing the test results.
o The laboratory must issue corrected reports promptly to the authorized person ordering the test or the individual utilizing the test results.
o The laboratory must maintain exact duplicates of the original and corrected report for two years.
Specialty/Subspecialty Requirements
Along with meeting the general requirements, laboratories must also meet the following specialty/subspecialty requirements
Bacteriological laboratories Specific Precautions
The laboratory must check positive and negative reactivity with control organisms:
• Each day of use for catalase, coagulase, beta-lactamase and oxidase reagents and DNA probes.
• Each week of use for Gram and acid-fast stains, bacitracin, optochin, ONPG, X and V discs or strips.
• Each month of use for antisera.
• Each week of use for X V discs or strips (with a positive control organism).
• Each new batch of media and each lot of antimicrobial discs before or concurrent with the initial use, using approved reference organisms. Zone sizes or minimum inhibitory concentration for reference organisms must be within established limits prior to reporting patient results. Each day of use, test appropriate control organisms to check the procedure.

Hospital Infection Control in Hematopoietic Stem Cell Transplant Recipients

Recently FDA approved stem cells transplantation as a line of therapy .
There were various suggestions for infections control guidelines among recepients of stem cells transplantations by CDC centre to review these valuablr guidelines you can read more
http://wwwnc.cdc.gov/eid/article/7/2/70-0263_article.htm

Chronic suppurative otitis media

When the eardrum has been perforated in an acute attack of otitis media and remains patent infection with the original pathogens may persist or repeated infections may be caused by secondary invaders such as S. aureus coliform bacilli, Pseudomonads and bacteroides. Swabs of the discharge in the external meatus should be cultured to guide the choice of antibiotics for systemic and topical therapy, but it must be borne in mind that such swabs are liable to be contaminated with commensal bacteria from the skin lining the meatus. These contaminants are mainly albus Staphylococci diplitheroid bacilli and saprophytic mycobacteria which should be ignored but may include s. aureus and coliform bacilli.
Otitis externa. Chronic inflammation of the skin of the external meatus, with irritation and discharge, may be caused by bacteria, particularly Pseudomonas aerugjnosa colifoon bacilli and S. aureus, or fungi, most commonly Candida or Aspergillus. A swab should be taken from the meatus and cultured aerobically on blood agar and MacConkey plates for the bacteria on a Sabouraud agar plate with a nystatin 50 Unit disk for 48h at 35-37°C for Candida and on a Sabouraud agar for 10 days at 28°C for Aspergillus . The results will guide the choice of drug for topical antibacterial or antifungal treatment.

Acute Otitis Media

This infection is usually caused by S. Pyogenes, Pneumococcus, H. infuenzae, Branhlamella catarrhalis or, in many cases, One of the respiratory tract viruses . The organism spreads to the middle ear via the Eustachian tube from the nasopharynx which is the primary site of infection As the eardrum remains intact, none of the infected exudates can be collected on an ear swab, through culture of a throat swab may give a provisional indication of the causal organism. Antibiotic therapy is urgently required to prevent a possible bacterial infection damaging the hearing mechanism and amoxycillin erythromycin or cotrimoxazole may be used when the causal organism is unknown. Amoxycillin is the drug of choice unless a β-lactamase-producing variety of H. influenzae is the cause, When the absence of a rapid response will indicate the need for a change of drug.
If the eardrum has ruptured spontaneously, or a myringotomy has been performed to relieve pressure exudates may be collected on a thin swab introduced carefully into the external meatus. It should be examined in a Gram film and by aerobic and anaembic Culture plates of heated blood agar and blood agar.

Laboratory Diagnosis of Cytomegalovirus

Laboratory diagnosis of HCMV infection
Samples
Various samples for detection of HCMV infection include body fluids, blood, polymorphnuclear leucocytes, tissues, etc. They should be rapidly transported to laboratory under sterile and aseptic conditions.


• Viral isolation

Human Diploid Fibroblast (HDF)
Cell cultures are obtained from the foreskin or from the embryonic lung tissues, have been used for conventional and shell vial methods for the isolation of HCMV. In conventional viral isolation methods, commonly used in earlier times, determination of viral replication is based on typical cytopathic effects (CPE) produced by HCMV. The time required for the development of CPE usually varies from 2 to 4 weeks, even up to six weeks.

Shell vial assay
One of the most commonly used rapid methods is the shell vial culture. In this method, for the improvement of absorption of the virus, the specimen is centrifuged onto the cell culture. Fibroblasts monolayers cultured in vials containing coverslips are used. These shell vial culture methods utilize indirect immunofluorescence to detect the immediate-early (IE) viral antigen after incubation of culture for one to three days
The spin amplification shell vial assay

The spin amplification shell vial assay has gained wide acceptance, as it is based on amplification of the virus in cell cultures after low speed centrifugation and detect viral antigen produced in the early replication of HCMV before appearance of CPE.

The isolation of HCMV from the blood or target organ specimen by cell culture methods has a high correlation with disease. However, it is relatively low sensitive in detection of the virus from blood samples compared to nucleic acid-based and antigenemia methods.

• Histological methods
Traditionally the recognition of cytomegalic inclusion bodies in histological specimens has been used for the diagnosis. In organ specific HCMV infection, such as HCMV pneumonitis or hepatitis, characteristic viral inclusions may be seen. The large inclusions are intranuclear and have a characteristic owl-eye appearance in haematoxylin and eosin stained tissue specimens. The positive results correlate well with active HCMV infection of the organ, but the sensitivity of the histopathological finding is relatively low.

• Immunofluoresense methods
Immunofluoresense staining with specific polyclonal or monoclonal antibodies against HCMV antigens has increased the sensitivity of the method compared to conventional staining. However, false-negative results may occur because of the focal and scarce distribution of HCMV positive cells in tissue samples.
Immunofluorescense (IF) either direct or indirect (IF) is a rapid diagnostic tool for viral diseases. The results are available within several hours after tissues obtained. The sensitivity of IF is improved when mixitures of monoclonal antibodies are used for detection of early and late antigens.
• Electron microscopy:
It could be used in detection of HCMV in urine, oral and other specimens. Positive results with almost all specimens that have infectivity titre >104 copies / ml.

• Serological methods

HCMV antibodies (IgM, Ig G)

Human cytomegalovirus infection is manifested by the production of IgG and IgM antibodies. Thus a diagnosis of HCMV infection can be obtained indirectly through serology. A variety of laboratory tests with different degrees of sensitivity have been described for the measurement of HCMV antibodies in human sera. The methods include complement fixation, indirect hemagglutination, latex agglutination, radioimmunoassay, immunofluorescence and enzyme immunoassay.

In enzyme-linked immunosorbent (ELISA) assays many different antigens have successfully been used as targets for detecting specific antibody production. The rise in serum antibody levels is an insensitive sign of actual HCMV infection in transplant patients.

The seroprevalence is high and the presence of IgG antibodies is only informative of the patient’s past history regarding HCMV infection. Furthermore, there is a time lag between primary infection and IgM antibody production (IgM level can remain undetectable because of delayed seroconversion owing to immunosupressive agents), and IgM antibodies can also persist for a long time after infection in some healthy individuals.

HCMV low avidity IgG

Primary HCMV infection can be detected by low avidity IgG. Also, in a study on renal transplant recipients there was an agreement between HCMV low avidity IgG and both direct Ag detection and polymerase chain reaction (PCR) in diagnosis of primary HCMV infection.

• Antigen detection(Antigenemia test)

The antigenemia assay, first described by professor The and his associates had a major advance in the diagnosis of HCMV infection in transplant patients. In this test, monoclonal antibodies to pp65 (the lower matrix phosphoprotein 65) are used for the direct immunostaining of blood polymorphonuclear leukocytes (PMNL).

It has been shown that the pp65 antigen in PMNLs is not a direct indication of virus replication in vivo, since the virus and viral material detected in PMNLs are transferred from other infected cells, e.g. endothelial cells, mainly by microfusion events.
The pp65 antigenemia assay consists of numerous steps, including isolation of PMNL, fixation, immunostaining, and microscopic evaluation and quantitation.

The disadvantage of the antigenemia assay is that the blood samples should be processed within a certain time, preferably within six hours, for optimal results.

It is still quite time-consuming and laborious, at least with large specimen numbers. The automation of the test is also difficult and the subjective evaluation of the infected leukocytes. Although there have been attempts to standardize the assay.

There are a number of various in-house and commercial modifications available. This makes comparison of the results between different centers difficult. The clinically significant threshold of the number of positive leukocytes seems to vary also among different types of transplant populations.

• Molecular methods


Nucleic acid amplification methods

Qualitative PCR has been proven to be more sensitive than antigenemia test or cell culture assay on the detection of HCMV infection.
Despite extreme sensitivity and specificity in detecting HCMV DNA, qualitative assays usually have a low positive predictive value for symptomatic infection, at least when peripheral blood leukocytes have been used as a specimen.
The sensitivity and specificity in detecting HCMV DNA is due to the capacity of these tests to detect HCMV DNA even in the case of latent infection. It has also been reported that the demonstration of HCMV DNA in plasma may correlate more closely with disease, and also with the antigenemia test, than that in leukocytes or whole blood.

The development and the availability of automated real-time instruments, have further simplified quantitative PCR assays and reduced the turnaround time needed for the test performance.


In real-time PCR the accumulation of the PCR products is monitored continuously during the PCR run, compared with the end-point measurements that quantitate the final PCR product. At present, several instruments are available for real-time PCR, in which the accumulation of the product is monitored by measuring the fluorescence in each cycle. The measured fluorescence is plotted against the cycle number.
There are various methods for the detection of PCR products during real-time PCR are available. These can be classified into amplicon sequence specific or non-specific detection methods. The most commonly used detection methods in the virus diagnostic assays are based on the use of specific fluorogenic oligoprobes .
These methods rely upon fluorescence resonance energy transfer (FRET), which is the interaction of two fluorescent dyes. TaqMan probes also called 5´ nuclease or hydrolysis oligoprobes, were the first ones used in special real-time instruments.
• Other nucleic acid amplification methods
Nucleic acid based sequence amplification (NASBA) is an isothermal amplification process which involves the coordinated activities of three different enzymes: ribonuclease H, reverse transcriptase and DNA-dependent RNA polymerase.

The final product of the process is a specific single stranded RNA, instead of the dsDNA of PCR. The high sensitivity in detecting HCMV infection has been shown by the NASBA assay with the (immediate early 1) IE-1 target.
There are many applications of the NASBA technique based on the detection of pp67 mRNA have been developed and the test application is also commercially available.

HCMV DNA has also been detected by a signal amplification method, Hybrid capture system, from blood samples of transplant patients. It is a solution hybridization assay that involves amplified detection instead of amplification of the desired nucleic acid fragment.

Prevention and treatment of HCMV disease

The pharmacologic agents used for HCMV prevention have evolved, from the use of acyclovir , immunoglobulins and IV and oral ganciclovir and valganciclovir.
Antiviral prophylaxis
One major strategy is antiviral prophylaxis, where antiviral drugs such as valganciclovir or oral ganciclovir are given to patients for at least 3 months after liver transplantation. However, antiviral prophylaxis is associated with delayed-onset HCMV disease, which typically occurs soon after completion of prophylaxis.