Hepatitis Viruses from A to B

Hepatitis B

HBV(DNA) from Hepadna virus
Incubation Peroid:
6 weeks-6 months
Mode Of Transmission:
Parenteral, sexual, vertical
Clinical Course:
Prolonged and more severe than A

Laboratory Diagnosis:
-Elevated ALT,AST from 10-100 folds Acute infection with resolution
-Viralantigens:
1) Surface antigen (HBsAg) is secreted in excess into the blood as 22 nm spheres and tubules. Its presence in serum indicates that virus replication is occurring in the liver
2) 'e' antigen (HBeAg) secreted protein is shed in small amounts into the blood. Its presence in serum indicates that a high level of viral replication is occurring in the liver
3) core antigen (HBcAg) core protein is not found in blood
-Antibody response:
1) Surface antibody (anti-HBs) becomes detectable late in convalescence, and indicates immunity following infection. It remains detectable for life and is not found in chronic carriers (see below).
2) e antibody (anti-HBe) becomes detectable as viral replication falls. It indicates low infectivity in a carrier.
3) Core IgM rises early in infection and indicates recent infection
4) Core IgG rises soon after IgM, and remains present for life in both chronic carriers as well as those who clear the infection. Its presence indicates exposure to HBV.

Figure: Acute Hepatitis B Infection

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Free Microbiological Books

Great site for free microbiological books
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Lectures in clinical microbiology: Chronic suppurative otitis media

Lectures in clinical microbiology: Chronic suppurative otitis media

Lectures in clinical microbiology: Control Procedures in Clinical Microbiology Laboratory

Lectures in clinical microbiology: Control Procedures in Clinical Microbiology Laboratory

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.

Cytomegalovirus Overview

Historical background

early the during newborns in described first was virus The as to referred infection systemic fatal a with of children urine the in cells large described reports multiple 20th century when mid 20 disease. In the cytomegalic inclusionth cell of development century with human cytomegalovirus (HCMV) of culture methods enabled propagation.

Virus structure and genome organization

Human cytomegalovirus (HCMV) belongs to the beta herpes virus family. Beta herpes viruses are more closely related to each other than to other herpes viruses with large genomic overlapping.
Beta herpes viruses are a subfamily of human herpes viruses that share architectural features of their virion, including a core containing a linear double-stranded DNA of 235-kb, an icosahedral capsid with 162 capsomeres. The capsid is surrounded by a proteinaceous tegument and an outer lipid envelope.

Virion proteins

Purified virions of human, murine, and simian CMV consists of at least 30 readily detected polypeptides with molecular weights ranging from 20 to over 200 kd, distributed in the capsid, tegument and envelope

Figure IX: Structure of Human cytomegalovirus (HCMV)

The capsid proteins

It is composed of seven proteins. The major capsid proteins (MCP) , minor capsid proteins (mCP), minor capsid binding protein (mC-BP), the smallest capsid protein (SCP) and three assembling protein related to proteins associate with capsids.

Tegument Structure and Function

The tegument in HCMV is located between the outer lipid membrane and the icosahedral protein capsid, which contains the viral genomic double-stranded DNA.

A tegument contains at least 25 proteins, of which are phosphorylated. The basic phosphoprotein 150 (pp150) and lower matrix protein 65 (pp65) are the most abundant proteins expressed during virus replication. The other prominent tegument proteins are pp28, pp71and huge tegument protein. Several transcriptional transactivator proteins have also been localized to the tegument.

The tegument proteins comprise more than half of the total proteins found within infectious virion. Tegument proteins are phosphorylated, but the significance of this phosphorylation and other post translational modifications to these proteins remains largely unexplored.

Envelope glycoprotein:
The envelope consists of two prominent glycoprotein complexes, a complex of dimers of glycoprotein B (gB), and a complex of the gH, gL, and gO. These glycoprotein complexes are assumed to play an important role in virus entry.

HCMV genome

HCMV DNA from strain AD 169 is linear, double stranded DNA molecule of 229 to 354 base pairs. It is divided into two unique regions termed unique short ( Us) and unique long (Ul). The Us is bounded by an inverted and a terminal repeat sequence. The polarity of genetic information in either of the unique regions can be inverted, leading to four isomeric forms of virion DNA.

The HCMV genome of AD 169 strains has been completely sequenced and contains 208 predicted proteins open reading frames (ORFs) of greater than 100 amino acid.

Figure : Structure of the HCMV genome of AD 169 strain: (UL = unique long region, US = unique short region, TRL = terminal repeat sequence long, TRS = terminal repeat sequence short, IRL = inverted repeat sequence long, IRS = inverted repeat sequence short). The most abundantly expressed viral genes at immediate early (IE) time are transcribed from the major IE locus (MIE), located in UL region (MIEP = major IE promotor). The primary transcript is differentially spliced to generate two major proteins, IE1 and IE2 ( Mocarski and Courcelle, 2001).

HCMV genotypes
Although most of the HCMV genome is highly conserved among the various HCMV strains, a subset of genes exhibits a high degree of variability. A high level of genetic heterogeneity usually occurs in a limited number of distinct genotypes.

Among the most variable genes are the viral envelope glycoprotein N (gN) and gO genes and the gene encoding the predicted membrane glycoprotein UL139.

Sequence analysis of highly polymorphic regions within these three genes allows the discrimination of seven distinct gN genotypes and eight distinct gO and UL139 genotypes. The existence of such a large number of distinct genotypes provides a useful tool for investigating HCMV population diversity within a host

Viral Growth Cycle

Human cytomegalovirus exhibits a restricted host range in cell culture. In vitro the most commonly used cells are primary differentiated human fibroblasts derived from skin or lung. HCMV can also replicate in endothelial, epithelial cells, smooth muscle cells and leukocytes.
The whole replication cycle of HCMV is slow, requiring approximately 48-72 hours. The cytopathic effect in response to HCMV is characteristically cell enlargement with intranuclear inclusions.

Attachment
Virions gain entry into a cell through a membrane fusion event involving the outer membrane of the cell and glycoproteins on the lipid envelope of virions. Once the fusion of these two membranes occurs, the DNA-containing protein capsid and the tegument proteins are released into the cell.





Penetration
Viral entry is the result of a cascade of interactions; HCMV attach epidermal growth factor receptor (EGFR) on permissive cells and other interactions between HCMV envelope glycoproteins and cellular integrins promote receptor clustering.
Following primary infection, these viruses, as well as other herpes viruses, maintain latency in the human host by integrating in the host cell chromosomes or by maintaining subclinical low level viral replication that is adequately controlled by a functioning immune system. The viruses may reactivate during immunosuppression in transplanted patients.
Viral gene expression
The gene expression pattern follows a similar cascade as used by other herpes viruses. During the lytic infection, viral immediate-early genes are expressed.

The expression of these genes results in the production of viral immediate-early (IE) proteins that modulate the host cell environment and stimulate the expression of viral early genes. These genes produce proteins that are responsible for replicating the double-stranded viral genomic DNA; after DNA replication. IE genes turn on the expression of viral late genes which are mainly structural components of the virion that assist in the assembly and release of newly formed viral particles.

A latent infection is characterized by the minimization of viral gene expression and the inhibition of the assembly and egression of new viral progeny .

DNA replication
After penetration, the capsid passes through the cytoplasm to a nuclear pore, the DNA is released. Viral DNA replication occurs along with the expression of the late genes that produce the gamma proteins which are mainly structural proteins.

Packing of viral DNA into newly formed empty capsids occurs in the nucleus. Maturation occurs by budding from the inner nuclear membrane.

HCMV Epidemiology and Transmission

Human cytomegalovirus infects humans of all ages. Acquisition of the virus usually occurs early in life, mainly during the first two decades. The great seroprevalence and early acquisition of the virus have been associated with lower socioeconomic circumstances, developing countries and over crowded populations.

Vertical transmission of HCMV may occur transplacentally, through contact with the virus in the maternal genital tract during birth and through mother's milk.

Horizontal transmission requires direct contact with infected body fluid, including saliva, urine, breast milk, semen and blood (Britt, 2008).
Following HCMV infection the virus is excreted in body fluids for months or even years and reactivation of the virus is common, especially in immunosuppressed patients ().

Increased risk for infection is clearly associated with sexual activity. Antibodies to HCMV are highly prevalent among male homosexual and women with sexually transmitted diseases.

Human cytomegalovirus is an important hazard of blood transfusion and organ transplantation. It is associated with other infections, with episodes of acute rejection and treatment with lymphocyte-depleting regimens that induce release of inflammatory cytokines.

Pathogenesis of HCMV infection

It has been suggested that epithelial cells of the upper alimentary, respiratory and genito-urinary tracts are the main sites of primary replication after the direct contact of infectious virus and mucosal surface.

Human cytomegalovirus viremia may last for several weeks during the primary infection. Hematogenous spread typically results in the infection of ductal epithelial cells at the initial site. HCMV can infect a wide range of tissues including salivary glands, gastrointestinal tract, lung, liver, brain, kidney and other tissues. Then it replicates in diverse cell types, including fibroblasts, epithelial cells, macrophages, vascular smooth muscle cells and endothelial cells.
The pathologic changes are minor in the immunocompetent host and infection is generally a symptomatic. In rare cases HCMV mononucleosis syndrome develops with potential complication including pneumonia, hepatitis, meningitis and autoantibody production (Percivalle et al., 1993).

In immunocompromised host, HCMV infection may lead serious effects;
• Gastrointestinal tract (GIT): causes GIT lesion and even perforation of the gut.
• Respiratory system: leads to severe problems mainly pneumonia).
• In kidney: leads to renal dysfunction and graft rejection In central nervous system: leads to inflammation and possible damage to retina and cochlea.
• Human cytomegalovirus is also suggested to infect the vascular wall and play a role in the development of atherosclerosis.

There are three morphological transforming regions called mtrI, mtrII, mtrIII which are found in the HCMV genome, in one study mtrII bind to tumor suppressor protein p53 and it down regulates p53 activated transcription, indicating its ability to alter p53 directed cellular regulatory mechanisms

In another study, HCMV causes reduction in human leucokytic antigen (HLA) class II molecules on HCMV infected macrophages that inhibits T- cell proliferation by different distinct pathways.

Infected cells may be multinucleated or bear intranuclear inclusions surrounded by a clear halo given rise to the term owl's eye appearance.

HCMV and immunity

• Role of cell mediated immunity
After the primary infection, long-term cellular and humoral immunity develops. The cell mediated immunity, including both natural killer cells and cytotoxic-T lymphocytes, plays an important role in the control of HCMV infection.

• Role of humoral immunity
Humoral immune response also develops but it has been suggested to be less important in the control of HCMV infection.

• HCMV and evasion the host immune system
The immune defence is unable to clear the virus completely from the host, and the virus remains latent lifelong within the host cell. The ability of the virus to evade the host immune system may be due to various HCMV gene products, which show sequence and functional relatedness to host proteins.

• HCMV latency and persistence
Human cytomegalovirus latency-associated transcripts have also been found in myeloid cells in both natural and experimental infection.

Tropism for different tissue targets is influenced by both viral and host determinants. The salivary glands are an important target for growth and dissemination of HCMV which replicate in salivary gland acinar epithelium).

Reactivation of a latent virus by allogeneic stimulation of peripheral blood mononuclear cells has been demonstrated. Differentiations of latently infected monocytes into macrophages by activation of the immune response lead to virus reactivation.

Cytokines activation and signal transduction pathways have been suggested to play an important role in reactivation of the virus. The differentiation state of the cell seems to be critical for the silencing of IE gene expression and thus for the maintenance of latency.

Clinical presentation of HCMV infection

A - Clinical syndromes
Human cytomegalovirus infects 60%-100% of humans, with primary HCMV infection occurring most commonly during the first 2 decades of life. In immunocompetent individuals, the infected individuals are mostly asymptomatic or may present with a benign febrile infectious mononucleosis-like illness.

In immunocompromised patients such as liver transplant recipients, clinical disease with high morbidity may develop and, in some cases, may lead to death.

• Infection in the normal host
Primary infection with HCMV is usually asymptomatic but in young adults, HCMV may cause mononucleosis-like clinical symptoms. Furthermore it represents the major infectious cause of birth defects.

Persistant fever, myalagia and non specific conistitutional symptoms. Infrequent complication of HCMV mononucleosis include pneumonia, hepatitis, aseptic meningitis, Guillian- Barre syndrome and variety of complications.

Post transfusion syndrome , after blood transfusion aquired HCMV infection usually self limited , but it may cause fever and hepatitis in hospitalized patients.

• Congenital infection
Maternal infection during pregnancy are classified primary and non-primary or recurrent infection. In primary infection during pregnancy result in a fetal transmission rate of 35% - 50%. In recurrent infection, fetal transmission rate range from 0.2% to 2% suggesting maternal immunity to HCMV.

Other factors that contribute in fetal transmission include younger maternal age, lower socioeconomic status and previous history of sexually transmitted diseases (STDs).

Fewer than 5% of congenitally infected infants develop symptoms during new born period ranging from intra-uterine growth retardation, jaundice, hepatosplenomegaly, mental retardation, centeral nervous system abnormalities.
• Delayed infection
In individuals who received antiviral prophylaxis, HCMV disease occurred 3 months to 6 months after completing antiviral prophylaxis; hence, the term “delayed-onset HCMV disease”.

Human cytomegalovirus (HCMV) hepatitis and other organs such as the central nervous system and the lungs may be infected, and present themselves through headache, delirium, changes in mental function, and cough and dyspnea, respectively.

• Infection in allograft recipients
The degree of immunosuppression of the recipient can be correlated with clinically significant HCMV infection. Seronegative transplant recipient of an organ from seropositive are at risk of primary infection and thus develop more serious disease than seopositive recipients of seropositive organs.

Allograft may provide a sequestered site for latency infected cells since MHC mismatch at these sites may prevent generation of major histocompitability complex (MHC) restricted virus specific cytotoxic T- cell responses.

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

The second strategy for HCMV disease prevention is pre-emptive therapy, which relies on a close virologic follow-up through serial blood markers (such as viral load or pp65 antigenemia) as the trigger for antiviral therapy, usually with intravenous ganciclovir or valganciclovir

The recommended agents and dose for prophylaxis are ganciclovir 5 mg/kg once daily for at least 3 months after transplantation, whereas in preemptive therapy, patients are closely monitored and given ganciclovir 5 mg/kg twice daily whenever CMV viremia is detected Ganciclovir-resistant HCMV is now emerging as an important complication of prolonged antiviral drug use after transplantation
The potential drugs for the treatment of multidrug resistant HCMV include the use of immunoglobulins and leflunomide. The potential clinical utility of maribavir in the treatment of resistant HCMV is highly anticipated

Transport of Microbiological specimens to the laboratory

Many pathogenic organisms don’t survive for long in clinical specimens kept at room temp. Examples include Gonococci, Haemophilus, Bacteroides, anaerobic cocci and most viruses.
On the other hand, some organisms contaminating specimens from the normal flora such as Coliform and Coagulase negative Staphylococci, may rapidly grow in specimen kept at room temp.
-Urine or sputum specimens should reach the laboratory within 2hours of collection when even possible. If delay are expected immediately inoculated into transport media.
-Transport media used:
Stuart’s transport media ----- for pus or swabs for bacterial culture when delays in transport.>1/2hour or when Neisseria infections are suspected. However the inoculated transport media should be sent to the laboratory within 4h.
The investigation of eye, genital tract is best carried at the bed side when suitable culture media are directly inoculated.
-Cerebrospinal fluid(CSF) not refrigerated since other wise Meningococci may rapidly die.
-Viral transport media is necessary for virus isolation, and also for Chlamydia isolation.
Specimens for virus isolation are kept at –70ºC till time of transferring the appropriate cell line which support growth of the possible virus or Chlamydia.

The role of the consultant clinical microbiologist:

A- Regular contact with clinical colleagues to ensure the appropriate investigation for the clinical conditions, and that good quality specimeas are sent
Organize the laboratory so that the relevant investigations are carried out reliably, safely and economically. Important results are communicated to colleagues and discussed.
B- As regard consultation on the investigation and management of patients with infection problems. Seeing patient on the words, temp. charts, drug shets. Etc., together with clinical colleagues
Discussing difficult clinical problems and management of out breaks of infectious disease
C-As regards Control of hospital infection: design implementation of policies on the use of antibiotics, isolation procedures, sterilization and disinfection
D- As regard teaching research: Education of medical staff about infections, the use of antibiotics, disinfectants. Etc.
-Research on epidemiology, diagnosis, treatment or prevention of infections.
Provision of essential clinical information:
The information routinely required in laboratory request: age, brief details of the main clinical condition , date of onset of the illness, antibiotic therapy, history of recent travel abroad, suspected source of infection.
Prior Discussion with microbiologist.
To achieve the best use of the laboratory certain types of investigation need to be discussed with the microbiologist. These include, assay of antibiotics the isolation of viruses, molecular biology tests, and the investigations of possible cross infection incidents

Yeasts Identifications

Blastoschizomyces
Candida
Cryptococcus
Loboaloboa
Malassezia
Rhodotorula
Saccharomyces
Trichosporon
Identification.
Yeast-like fungi may be basidiomycetes, such as Cryptococcus neoformans or ascomycetes such as Candida albicans.
1. Ensure that you start with a pure culture; streak for single colony isolation if necessary.
2. Germ Tube Test: lightly inoculated 5 ml of serum, containing 0.5% glucose and incubated at 35oC for 2-3 hours.
Positive = Candida albicans or Candida dubliniensis.
Negative or from HIV positive patient = perform assimilation tests.
3. For the identification of germ tube negative yeasts, morphological (Dalmau plate culture), physiological and biochemical tests are essential.
Dalmau Plate Culture: To set up a yeast morphology plate, dip a flamed sterilized straight wire into a light inoculum (sterile distilled water suspension) and then lightly scratch the wire into the surface of a cornmeal/tween 80, rice/tween 80 or yeast morphology agar plate, then place a flamed coverslip onto the agar surface covering the scratches. Dalmau morphology plates are examined in situ using the lower power of a microscope for the presence of pseudohyphae which may take up to 4-5 days at 26oC to develop. C. albicans also produces characteristic large, round, terminal, thick-walled vesicles (often called chlamydoconidia). The key features to remember are to use a light inoculum and to scratch the surface of the agar with the wire when inoculating.
Physiological and biochemical tests: including fermentation and assimilation studies should be performed. Reliable commercially available yeast identification kits are the API 20C AUX, ATB 32C, MicroScan and Vitek systems
Identification of Common Dermatophytes.
Microscopic morphology of the micro and/or macroconidia is the most reliable identification character, but you need a good slide preparation and you may need to stimulate sporulation in some strains. Culture characteristics such as surface texture, topography and pigmentation are variable and are therefore the least reliable criteria for identification. Clinical information such as the site, appearance of the lesion, geographic location, travel history, animal contacts and race is also important, especially in identifying rare non-sporulation species like M. audouini, T. concentricum and T schoenleinii etc. Note: mating experiments are not practical for the clinical mycology laboratory.
Three genera are recognized:
Epidermophyton:
Smooth thin-walled Macroconidia only present, no microconidia, colonies a green-brown to khaki colour.
Microsporum:
Macroconidia with rough walls present, microconidia may also be present.
Trichophyton:
Microconidia present, smooth-walled macroconidia may or may not be present.
Lactophenol Cotton Blue.
For the staining and microscopic identification of fungi

Method
This stain is prepared over two days.
1. On the first day, dissolve the Cotton Blue in the distilled water. Leave overnight to eliminate insoluble dye.
2. On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. place on magnetic stirrer until the phenol is dissolved.
3. Add the glycerol.
4. Filter the Cotton Blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at room temperature.
Sabouraud's Dextrose Agar for Dermatophytes
Sabouraud's Dextrose Agar with Cycloheximide, Chloramphenicol, Gentamicin and Yeast Extract.
For the primary isolation and cultivation of dermatophytes.

Method:
1. Soak all ingredients, except Gentamicin, in 100 ml water.
2. Boil remaining water, add to soaking ingredients, and bring to boil to dissolve, stirring well to prevent from charring.
3. Add the Gentamicin. Mix well.
4. Dispense for slopes if required.
5. Autoclave at 121C for 10 minutes. Remove and slope, or pour for plates as required.

Sampling for FUNGAL Infections and Culture

Skin Scrapings and Swabs
In patients with suspected tinea or ringworm any ointments or other local applications present should first be removed with an alcohol wipe. Using a blunt scalpel, tweezers, or a bone curette, firmly scrape the lesion, particularly at the advancing border. A bone curette is safe and useful for collecting specimens from babies, young children and awkward sites such as interdigital spaces. If multiple lesions are present choose the most recent for scrapings as old loose scale is often not satisfactory. Any small vellus hairs when present within the lesions should be epilated. The tops of any fresh vesicles should be removed as the fungus is often plentiful in the roof of the vesicle.
In patients with suspected candidiasis the young "satellite" lesions which have not undergone exfoliation are more likely to yield positive results if they are present. Otherwise the advancing scaly border should be scraped. When lesions in the flexures are moist and very inflamed it is more satisfactory and less painful to roll a moistened swab firmly over the surface.
In patients with suspected cutaneous manifestations of systemic pathogens scrap the lesions with a bone curette or blunt scalpel as for tinea. A biopsy may be required in some cases.
NOTE: Following the collection of skin scales all scraped lesions should be firmly rubbed with a swab moistened in BHI broth.
Skin Scrapings, nail scrapings and epilated hairs where tinea is the provisional diagnosis:
1. Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimen onto two slopes containing cycloheximide (actidione) i.e. one DERMASEL agar slope and one LACTRITMEL agar slope also containing chloramphenicol, gentamicin and incubate cultures at 26C. Maintain cultures for 4 weeks.
3. Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.
Skin scrapings and swabs where candidiasis is the provisional diagnosis:
A. Skin scrapings:
1. Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar slopes containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks.
B. Skin Swabs:
1. Smear swab onto heat sterilized glass slide for Gram stain.
2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks.
3. Where secondary bacterial infection is suspected, and separate swabs for routine bacteriology were not collected, the swab should first be inoculated onto a blood agar plate, followed by the Sabouraud's agar containing the antibiotics and then placed into Brain Heart Infusion Broth. All cultures should be incubated at 35C. Maintain cultures for 4 weeks.
NOTE: Where a dermatophyte is suspected or to be excluded a Sabouraud's agar slope containing cycloheximide and incubated at 26C may be included.
Scrapings from the groin, feet or nails where either a dermatophyte or Candida species may be isolated. This includes the possibility of a non-dermatophyte onychomycosis.
1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide (as for Candida) and incubate at 26C. Maintain cultures for 4 weeks.
3. Inoculate specimen onto a DERMASEL agar slope containing cycloheximide (actidione), chloramphenicol and gentamicin and incubate cultures at 26C. Maintain cultures for 4 weeks.
4. Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.
Skin scrapings from patients with suspected pityriasis versicolor:
1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy along with the cellotape stripping taken at the time of collection.
2. Inoculate scrapings onto an DIXON'S agar slope for isolation of Malassezia furfur and incubate cultures at 26C. Maintain cultures for 4 weeks.
3. Inoculate specimen onto Sabouraud's dextrose agar with chloramphenicol and gentainicin but NO cycloheximide (actidione) and incubate cultures at 26C. To exclude other yeasts like Candida. Maintain cultures for 4 weeks.
4. If dermatophytes are to be excluded also inoculate onto DERMASEL agar slope and incubate cultures at 26C. Maintain cultures for 4 weeks.
Skin scrapings from patients where a systemic pathogen is suspected:
1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto:
(a) Sabouraud's dextrose agar with chloramphenicol and gentamicin but NO cycloheximide (actidione) and incubate duplicate cultures at 26C and 35C; and
(b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Sputum, Bronchial Washings and Throat Swabs
Many opportunistic mycoses have a pulmonary origin following the inhalation of fungal propagules. Bronchial washings and sputa should be collected upon rising in the morning as overnight incubation and growth of fungi in the lungs will increase the likelihood of isolating pathogenic fungi. Patients should not eat before specimen collection. Twenty-four hour samples are unacceptable because they become overgrown with bacteria and fungal contaminants. It should also be stressed that bronchial washings and sputa will usually be contaminated with throat flora. For this reason interpretation of results may be difficult from poor quality specimens.
Throat specimens are obtained by rolling a moist sterile swab over the affected area. However, if Candida is suspected the affected area will need to be scraped with a sterile tongue depressor.
All specimens must be sent to the laboratory and processed as soon as possible, a delay of longer than two hours at room temperature may impede the detection of some fungi. Store at 4C if short delays in processing are anticipated.
Unless it is already sufficiently fluid to pipette with a Pasteur pipette, sputa may need to be emulsified by shaking with l2-20 sterile glass beads and about 3-5ml of sterile distilled water, depending on the volume of the original specimen. Any bits of blood, pus or necrotic material should be plated directly onto media.
(1) Make wet mount preparations in KOH (l drop) and Gram stained smears (l drop) of all suspicious areas. The PAS stain may be necessary if the KOH preparation is unsatisfactory.
(2) Inoculate sample onto:
(a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and 35C; and
(b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Blood and Bone Marrow
The laboratory should be informed by the physician if fungal septicemia is suspected because special media are necessary for the optimum recovery of fungi. Numerous blood culture systems are available; however all systems must be vented to atmospheric air and incubated at 30C to maximize the rate and time of recovery of fungal organisms.
Aseptically collect 10 ml of blood and prepare several smears for Giemsa, Gram and PAS staining. Culture the remaining specimen by one or more of the following methods. With bone marrow aspirates the initial material is generally used for making smears for Giemsa staining, the remaining 3-5 ml of marrow and blood may be cultured on the media listed below.
1. Direct culture method: Inoculate 0.5-1.0 ml of buffy coat, prepared by centrifuging 5-10 ml of blood, onto the surface of the media listed below. This inoculum can then be spread over the surface of the agar with a sterile inoculating loop and the plate incubated aerobically at 30C. This method is suitable for small low volume laboratories where there are few requests for fungal blood cultures. Cultures should be maintained for 4 weeks.
2. Biphasic culture bottle: The recovery of fungi from blood may be enhanced by using a biphasic bottle containing a slant of brain heart infusion agar and 60-100 ml of BHI broth. A ratio of 1:10 to 1:20 (blood to broth) is recommended, a minimum of 5.0 ml of blood is required for each culture bottle. The biphasic culture bottle is kept vented and is tilted daily to allow broth to flow over the agar surface. These cultures must be carefully checked daily for growth. Because fungi will not turn the broth very cloudy it is imperative to frequently Gram stain the bottle contents to detect fungal elements. Cultures should be incubated at 30C and maintained for 4 weeks.
3. Membrane filter technique: This is a superior technique to the vented biphasic blood bottle used to concentrate and culture specimens of blood and CSF. Briefly, specimens are treated sequentially with Triton-X and sodium carbonate solutions to lyse blood cells and then filtered by vacuum through a 0.45 um membrane. This membrane is then placed onto the media listed below.
4. Lysis centrifugation isolator system: The Wampole Isolator system has been found to significantly improve the recovery of fungi from blood and is strongly recommend by Koneman and Roberts (1985) as the method of choice for processing blood cultures from patients with suspected fungal septicemia. The Isolator utilizes a tube that contains components that lyse leukocytes and erythrocytes and also inactivate plasma complement and certain antibiotics. Once lysed, the cells release the microorganisms contained within them, and the centrifugation step in the procedure serves to concentrate the organisms in the blood sample. This concentrate is then inoculated onto the surface of appropriate culture media listed below. Ten milliliters of blood are required for each tube and cultures should be incubated at 30oC and maintained for 4 weeks.
5. Bactec: Bactec have produced a special fungal media (BACTEC Fungal Medium) for enhanced fungal blood culture using their non-radiometric (NR) instruments. Once again, blood cells are lysed by the medium to enhance recovery of fungi. Note antimicrobials have also been added to limit the growth of bacteria.
Primary isolation media for blood and bone marrow culture: (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26oC and 35oC; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Note: Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26C for 4 weeks to exclude the presence of a slow growing fungus
Urine should be collected first thing in the morning after overnight incubation in the bladder. A midstream clean catch or catheterized specimen is best, as this minimizes the presence of genital flora. Do not use urine from a collection bag or bed pan. Twenty-four hour urine samples are unacceptable.
Yeasts recovered from routine urine bacteriology cultures of catheterized urine or urine obtained by sterile procedure should be identified and reported regardless of colony count. However, the isolation of yeasts from clean catch specimens must be interpreted with caution and is not significant without additional support from other clinical and laboratory investigations.
Note: Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26C for 4 weeks to exclude the presence of a slow growing fungus.
Urine samples must be processed as soon as possible, a delay of longer than two hours at room temperature may impede the detection of some fungi. Store at 4oC if short delays in processing are anticipated.
(1) Centrifuge the urine for 10-15 minutes at 2000 rpm. Decant the supernatant and pool the sediment if necessary.
(2) Prepare a direct smear of the sediment in KOH for direct microscopy. Note PAS, Gram or India ink preparations may also be helpful.
(3) Inoculate 0.05-0.1 ml of the sediment onto Sabouraud's agar with gentamicin and chloramphenicol and incubate duplicate cultures at 26C & 35C. Maintain cultures for 4 weeks.
Cerebrospinal Fluid (CSF):
Three to five milliliters of CFS is optimal for fungal investigation, however lesser volumes are often received and should be processed. CSF specimens should be transported to the laboratory as soon as possible and processed promptly. If there is a delay do not refrigerate the samples, rather they should be left at room temperature or incubated at 30C. The specimen should be centrifuged. Keep the supernatant for cryptococcal antigen testing and process the sediment as follows.
(1) For direct microscopy use 1 drop of the sediment to make an India ink mount.
(2) Resuspend the remaining sediment in 1-2 ml of CSF and inoculate onto; (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and 35C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for at least 4 weeks.
Note: Cultures from patients undergoing treatment for cryptococcal meningitis should be maintained for 3 months, so that dormant viable cells, which do not start to grow until after a one month period, will not be missed.

Microbiology Laboratory Quality Control

General Requirements
For moderately and highly complex tests, the laboratory must:
􀁺 Follow the manufacturer's instructions.
􀁺 Have a procedure manual describing the process of the tests
and reporting patient test results.
􀁺 Perform and document calibration procedures or check
calibration at least once every six months.
􀁺 Perform and document control procedures using at least two levels of controls each day of testing.
􀁺 Perform and document applicable specialty and subspecialty control procedures.
􀁺 Perform and document remedial action taken when problems or errors are identified.
􀁺 Maintain records of all quality control activities for two years (five years for immunohematology).
Facilities
The laboratory must have space and environmental conditions necessary for conducting the services offered. This includes being constructed, arranged and maintained to ensure the space, ventilation and utilities necessary for conducting all phases of testing.
Safety precautions must be established, posted and observed to ensure protection from physical, chemical, biochemical and electrical hazards and biohazardous materials.
Test Methods, Equipment, Instruments, Reagents, Materials and Supplies
The laboratory must utilize test methods, equipment, instruments, reagents, materials and supplies that provide accurate and reliable test results and reports.
Requirements include
-Utilize appropriate and sufficient equipment, instruments, reagents, materials and supplies for the type and volume of testing performed and for the assurance of quality throughout the testing; and, test result reporting, including (as applicable): -Selecting methodologies and equipment and performing testing in a manner that provides test results within the laboratory's stated performance specifications for each method
-Water quality
-Temperature
-Humidity
-Protection of equipment and instrumentation from fluctuations and interruptions in electrical current that adversely affect results and reports
Document remedial actions implemented to correct conditions that fail to meet criteria
-Label reagents, solutions, culture media, control materials, calibration materials and other supplies including identification for:
Storage requirements-
-Identity, and when pertinent, titer, strength or concentration
-Preparation and expiration date Other appropriate information
-Prepare, store and handle reagents, solutions, culture media, control materials, calibration materials and other supplies in a manner to ensure:
- Items are not used when they have exceeded their expiration date, have deteriorated or are of sub-standard quality.
-Components of reagent kits of different lot numbers are not interchanged unless otherwise specified by the manufacturer
Control procedures
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.

Automation in Micrbiology (Part II)

Automated non radiometeric methods
• MGIT( mycobactrium growth indicator tube)

-- modified middle brook 7H9 with florescent quenching based oxygen.

-- with the utilization of oxygen during growth florescence is released and detected.

-- either fully automated or manual reading.


b) Bactialert 3D system

Figure our Bactialert in Mansoura University Hospital
c) VersaTrek culture system

2- automated system for biochemical reactions
1st API:
 A plastic strip holding mini-test tubes is inoculated with a saline suspension of a pure culture reacts with dessicated medium in each tube, sometimes oil is added to the surface for anerobes.

 incubation in a humidity chamber for 18-24 hours at 37°C, the color reactions are read.

 The reading are converted to a seven-digit code which is called the Analytical profile index(API)
A.Uninoculated control

B.Red slant and red butt, no black color= no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced

C.Red slant and black butt= no lactose or sucrose fermentation, H2S has been produced

D.Red slant with yellow butt= no lactose or sucrose fermentation, lactose is fermented, no H2S has been produced

E.Yellow slant, yellow butt and black coloration= Lactose, sucrose and glucose fermented, and H2S has been produced

F.Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been produced but gas has been produced

G.Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose,sucrose glucose,


2nd microscan
3rd Vitek 2
4th Rapid-ANA II

 Depend on the presence of pre-existing bacterial enzymes and so the rapid detection.

 The results are avaliable within 4-6 hrs.

Microscan

Figure Microscan in Mansoura University hospital

Figure Microscan® Gram Positive Breakpoint Combo panels Type 20

Vitek 2
3-Automated susceptibility testing methods
 A series of commercially available automated and semi-automated methods

 Most of the methods combine bacterial identification and susceptibility testing reagents in a single panel or card.

 The goal of the automated methods is to reduce the time necessary to produce accurate identification and susceptibility test result.

 Indeed, results may be available for some bacterial species in as little as 6 hrs.

 Overall, automated systems work well, although they have traditionally shown problems with certain resistance phenotypes, including oxacillin-resistant S. aureus isolates and Pseudomonas aergnosa.

 Finally, the detection of resistance due to induction of B-lactamases
( Enterobacteriaceae), and third-generation cephalosporins), has been addressed in many of the automated systems by use of low concentrations of B-lactams as screening tests.
Inhibition Zone Test
(synbiosis)
AST using turbidimetery

Automated Systems In Microbiology

Types of Automated systems used in microbiology

Culture methods
Biochemical identification systems
Antibiotic susceptibility
Antigen/antibody detection
Nucleic acid identification
1-Automated systems for culture

Including :

Bactec 9050
VersaTrek
Bactialert 3D system


Bactec 9050

Using floresent detector.

During growth of bacteria co2 is produced, and begin to diffuse to release hydrogen which enhance emission of florescence detected with special detectors .

Positive samples could be detected within 6 hrs versus 10 days in case of septicemia.
VersaTrek

Depend on consumption/production of multiple gases (co2,H2,O2).

This cause changes in the pressure of the head space which could be detected.

Figure: Our BACTEC System in MUCH
Bactialert 3D system
There is a PH senstive membrane in the bottom of the bottles.

When the organism grow, co2 is released causing changes in PH

This change in PH cause colour change from gray to orange or yellow.

How it works?
Bactialert 3D system
There is a PH senstive membrane in the bottom of the bottles.

When the organism grow, co2 is released causing changes in PH

This change in PH cause colour change from gray to orange or yellow.

How it works?
Culturing techniques for mycobactrium

All techniques depend on liquid media middle brook 7H.

1st Bactec 460

is a radiometeric method using radioactive carbon 14.

The organism utilize carbon to release radioactive co2 into the headspace of the glass vial.

Amount of co2 is proportional to growth (growth index).

Detection within 9-14 days versus 3-6 weeks.

To be continued>>>

UPPER RESPIRATORY INFECTIONS

The commonest respiratory infections are localized in the oropharynx, nasopharynx and nasal cavity, causing sore throat, nasal discharge and often fever, but the throat pathogens may also spread to infect the larynx, causing hoarseness, the middle ear, causing otitis media with earache, a paranasal sinus, causing sinusitis with pain in the face or head, and the eye, causing conjunctivitis or keratitis. The upper respiratory tract may also be involved in wider respiratory or generalized infections such as whooping cough, influenza, measles and infectious mononucleosis.
In most cases the primary infection is viral, though the causal virus is generally not demonstrated, and there is often concomitant carriage or secondary infection with one of the potential bacterial pathogens commonly present in the nasopharynx, e.g. Pneumococcus, Haemophilus infiuenzae (H. infiuenzae). Staphylococcus aureus and Streptococcus pyogenes (S.pyogenes). Drug-resistant Coliform bacilli or yeasts may come to dominate the throat flora in patients receiving antibiotics, but their presence is generally of little pathological significance.
Streptococcal pharyngitis
The only common primary bacterial cause of sore throat is S. pyogenes, which is found in about 30% of cases of pharyngitis, with or without tonsillitis. Its detection is the main purpose of the bacteriological examination of throat swabs, for it is the only common throat pathogen for which antibiotic therapy is clearly indicated. When it is thought or shown to be present, benzyipenicillin and procaine penicillin should be given by intramuscular injection and followed by phenoxymethyl penicillin given orally for 7-10 days; commonly, the injections are omitted. Erythromycin should be given to patients allergic to the penicillins. Effective therapy should cause rapid amelioration of symptoms, e.g. within 24-48 h, and prevent serious complications such as otitis media and rheumatic fever. Streptococcal pharyngitis cannot be distinguished clinically from viral pharyngitis, so whenever practicable throat swab examinations should be made on patients with sore throat.
In only a few cases are there clinical indications requiring the examination of throat swabs for other pathogens, such as the diphtheria bacillus , Vincent's organisms. Candida or Gonococcus and in the absence of such indications swabs from pharyngitis should be examined only for S.pyogenes. If the presence of commensal nasopharyngeal residents like H. infiuenzae. Pneumococcus, S. aureus and coli-form bacilli is reported to the physician, he may be induced to give inappropriate antibiotic therapy.
Method of Throat swabs. A plain, albumen-coated or charcoal-coated cotton-wool swab should be used to collect as much exudates as possible from the tonsils. posterior pharyngeal wall and any other area that is inflamed or bears exudates. If the patient permits, the swab should be rubbed with rotation over one tonsillar area, then the arch of the soft palate and uvula, the other tonsillar area, and finally the posterior pharyngeal wall. An adequate view of the throat should be ensured by good lighting and the use of a disposable wooden spatula to pull outwards and so depress the tongue. The swab should be replaced in its tube with care not to soil the rim. If it cannot be delivered to the laboratory within about 1 h, it should be placed in a refrigerator at 4°C until delivery or, preferably, it should be submitted in a tube of transport medium.
Culture of Throat swab. In the laboratory the swab should be rubbed, while being rotated, over large ‘well’ areas, about one-third of the surface on each of two blood agar plates, and the wells should be streaked out with a loop over the remainder of the plate. The plates should be incubated at 37°C for 18-24 h, one in air plus 5-10% CO2. the other anaerobically in nitrogen or hydrogen plus 5-10% CO2. It is advantageous, before incubation, to place a 6 mm disk containing 1 unit of benzylpenicillin on the well area of one plate and a disk containing 0.1 units of bacitracin on that of the other.
Next day, colonies of S. pyogenes are recognized by their zones of β-haemolysis, larger and clearer on the anaerobic than the aerobic plate, and their sensitivity to both penicillin (zone diameter >16 mm) and bacitracin (zone >12 mm). Haemolytic Haemophili. which have streptococcus like, colonies, give stronger haemolysis on the aerobic than the anaerobic plate and are resistant to penicillin. The results of these primary sensitivity tests make possible the provisional identification of S. pyogenes, and its immediate reporting to the physician. even when the streptococci and their haemolysis are confined to the confluent mixed growth in the well of the plate and separate β- haemolysis colonies are not available for testing.
The bacitracin test fails to identify rare strains of S. pyogenes that are bacitracin-resistant and misidentifies rare strains of other streptococci that are bacitracin-sensitive. If, therefore well separated β-haemolytic colonies are present on the streaked-out area of the plate. they should be picked and their Lancefield group determined by a rapid co-agglutination or precipitation test . When separate colonies are not present. it is necessary to repeat the confluent (β-haemolytic growth to obtain a pure culture for grouping, but a report to the physician should not be delayed until the results of this confirmatory test are available.
Quantitative Culture. Note and report the relative abundance of S.pyogenes colonies in the primary plate culture, for the organism is more likely to have a pathogenic role when it is numerous (e.g. >100 colonies/plate) than when, it is scanty. An appreciable proportion of healthy persons, e.g. 1-10% of adults and up to 20 or 30% of children, carry the streptococcus in the throat, apparently without harm, and the organism will be detected in a throat swab when a carrier develops a sore throat due to some other pathogen, such as an undetected virus. Such a finding could lead to a misdiagnosis, and if the streptococci are scanty the possibility that they are not the cause of the pharyngitis should be borne in mind.
In a study of the value of standardized quantitative culture, it was found that large numbers of S. pyogenes in throat swabs from 71% of children with streptococcal pharyngitis, but in swabs from only 10% of well children who were throat carriers of the organism. But the numbers of streptococcal colonies on the culture plate are greatly influenced by the efficacy of the procedures of swabbing the throat and transfer to the plate, so that they may not fairly reflect the number of the organisms in vivo . As these procedures cannot be rigidly standardized. account should also be taken of the number of S. pyogenes colonies relative to the number of colonies of throat commensal bacteria in the culture. Although a scanty growth of the streptococcus is likely to be due to harmless throat carriage, the uncertainty of the quantitative distinction makes it advisable that in all cases the organism’s presence should be reported and antibiotics given to eradicate it.
Repots may be given as ‘Many’, Few’ or No S. pyogenes found in culture’. When none is found. it is advisable to add to the report a statemert that ‘Other pathogens, including viruses were not sought’. If a negative result is reported only as ‘No pathogens isolated’, the physician may think that the swab has been examined for viruses, Mycoplasmas, Chlamydias, Diphtheria bacilli, Vincent’s organisms, Gonococci and other rarer throat pathogens.
Anti-streptolySin-O (ASO) titer. In cases of suspected streptococcal infection, e.g. acute rheumatic fever, where throat and other cultures have failed to reveal the organism, the patient’s blood serum should be tested for its content of antibodies to streptolysin-O. In such infections there is usually a steep rise of ASO titer to values well in excess of 200 Todd unit/ml after 2-4 weeks. If the titer is not raised, the patient’s illness is unlikely to be rheumatic fever. A commercial kit (Rapitex ASL) is available from Behring for rapid testing with a suspension al latex particles sensitized with streptolysin-O.
Other throat infections
Haemolytic streptococci other than S. pyogenes are often present in the throat as harmless commensals, but those of groups C and G occasionally, and B rarely, cause pharyngitis. If their presence in large numbers suggests they may have a pathogenic in the patient, their presence and antibiotic sensitivities should be reported to the physician.
Haemophilus influenzae. As haemophili are carried as commensals in the throat in a large proportion of adults and children, their finding in a throat swab should generally be ignored. There are, however, certain circumstances in which a search should be made for them and their presence regarded as possibly significant. Thus, H. influenzae of capsule serotype b is a fairly common pathogen in young children, especially under the age of 4, in whom it may cause pharyngitis,tracheo-laryngo-epiglottitis (croup). bronchopneumonia, bacteraemia and meningitis, and in such cases it should be sought in throat swab cultures. Moreover, in children with suspected bronchitis or pneumonia, it is often difficult to obtain a satisfactory specimen of sputum, and it may then be helpful to examine a throat swab for the presence of Haemophilus and other potential lung pathogens and determine their drug sensitivities. The physician should, however, be warned that though the pathogen in the throat may also be present in the lower respiratory tract, it is more probably confined to carriage in the throat. It may be difficult or hazardous to collect a throat swab from a child with croup, in which case an attempt should be made to demonstrate the pathogen in blood culture, as should also be done in suspected pneumonia and meningitis.
When Haemophilus is to be sought, the throat swab should be inoculated on to a heated-blood agar or Fildes agar plate as well as on to blood agar, and that plate incubated aerobically. A 2µg amoxycillin or ampicillin disk may be placed on the well of the plate so that both the presence and sensitivity of the haemophilus may be reported next day. Until the ampicillin sensitivity is known, the drug of choice for the initial treatment of severe haemophilus infections is chloramphenicol, but its prolonged administration may be dangerous.
Diphtheria. In countries where diphtheria is even moderately common, all swabs from sore throats should be cultured on a selective tellurite indium for Corynebacterium diphtheriae and C. Ulcerans as well as on blood agar S. pyogenes.
Eleks test is used to detect exotoxin production from the isolated bacteria. It is composed of filter paper impregnated in antitoxin and put against the susbected culture.Precipitation lines will be formed around the filter paper
In communities where artificial immunization has made diphtheria rare, the chance of making a positive finding may be too low to justify the large expenditure of labor and materials in routinely setting up the extra cultures. In that case, reliance must be placed on the physician to indicate the few cases in which the possibility of diphtheria has been suggested by the presence of membrane in the throat, extreme constitutional upset or nerve paralyses, and which therefore require examination of the swab by the these methods.
Vincent’s infection. A foetid, ulcerative inflammation of the throat (Vincent’s angina) or gums (gingivitis) is occasionally caused by a combined infection with Vincent’s spirochaetes and anaerobic fusiform bacilli. When the clinical findings suggest the condition, a swab from the affected areas should be examined in a Gram smear. The presence of many Gram-negative Spirochaetes and Fusiform bacilli, e.g. at least two of each per field well filled with pus cells and debris, may be reported as ‘Many Vincent’s organisms in film’. Small numbers of such organisms may be present in the healthy mouth and throat, and should be ignored.
Gonococcal pharyngitis. This condition should be suspected in promiscuous persons who engage in oral intercourse and a swab should be examined for gonococcus by culture on selective medium.
Candidiasis (thrush). In newborn babies and debilitated elderly persons, infection with Candida albicans may cause an acute inflammation with plaques of soft white exudates in the mouth and throat. in patients with these manifestations, a swab taken from the lesions should be examined for the fungus. An aerobic blood agar plate may show the small opaque white colonies of Candida, typically with short pointed ‘rootlets’ projecting from their margins, hut their growth may be slow and incubation at 35-37°C may have to be continued for 48h before they become recognizable. When an examination for candida is indicated. it is best to inoculate the swab on to a plate of Sabouraud agar as well as on to blood agar. A 50 unit nystatin disk and a 20µg amphotericin disk should be placed on the ‘well’ of the Sabouraud plate. Growth of candida sensitive to the antifungal drugs may then be observed and reported after 24 or 48h. and later be subjected to confirmatory tests. The presence of small numbers of yeasts in material from the mouth or throat may reflect selection and opportunistic colonization during antibiotic therapy. In such a case, further antibiotic therapy may be contraindicated.
Viral infections. Several viruses may cause an exudative pharyngitis resembling that caused by Streptococcus pyogenes. One that commonly. does so is the virus of infectious mononucleosis, a condition which may be diagnosed by the demonstration of a lymphocytosis and atypical lymphocytes in a blood film and that of heterophile antibodies in a Paul-Bunnell test on the patient’s serum. Diagnostic tests are usually not attempted for other viral infections of the throat unless the identification is required for epidemiological purposes, when viral culture and serological diagnosis may be attempted.