Clinical Mycology

Fungi constitute a group of non motile, eukaryotic organisms that have definite cell walls, devoid of chlorophyll and reproduce by means of spores either sexual or a sexual. Classification: Fungi may take the following forms 1-Yeast: uni cellular spherical or ovoid in shape such as Candida species. 2-Mold: multicellular such as dermatophytes. 3- Dimorphism: is a character of some pathogenic fungi which grow as molds in natural environment and in lab. Fungal Infections: They are classified into: cutaneous, sub-cutaneous, deep and opportunistic mycosis Type Causative Fungus Infection A) Cutaneous Malassezia Tinea versicolor Dermatophytes (Epidermophyton, Trichophyton,Microsporum) Ring worm of skin, nails and hair. Subcutaneous Sporothrix Sporotrichosis of lymph vessels and lymph nodes Several genera Mycetoma (Madura foot) chronic granuloma discharging pus especially of leg and foot Systemic Histoplasma Pulmonary or disseminated histoplasmosis Coccoidiodes Pulmonary or Erythema Nodosum Blastomyces Pulmonary or disseminated Paracoccoidiodes Pulmonary, disseminated Opportunistic Candida Candidiasis: oral thrush, vaginitis or dissiminated Aspergillus Aspergillosis: pulmonary, aspergilloma, toxicosis (ASP, Flavus produce aflatoxin which is hepatocarcinogen. Cryptococcus Cryptococcal meningitis or pulmonary cryptococcus Mucor Mucor mycosis of bllod vessels esp. of paranasal sinus, lung, gut. Diabetic ketoacidosis, organ transplant and leukemic patients are mostly susceptible. Laboratory Diagnosis: A) Collection of samples: according to site of infection, cutaneous (hair, nail, skin), subcutaneous (abscess, sinus, fistula), systemic (blood,sputum, CSF, bone marrow, urine, faeces). B) Diagnostic methods: 1-Direct microscopic examination using KOH-indian ink,Giemsa –periodic acid Schiff. 2-Fungal culture: Common media is Sabouraud s agar. 3-Direct antigen detection by immunoelectrophoresis or latex. 4- Serology useful for diagnosis and prognosis of systemic fungal infections.

Diagnoses of Infectious Diseases

Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods: - Conventional Methods Laboratory tests may identify organisms either directly microscopically by stains, growing organisms by cultural techniques and/or detecting their antigens or indirectly by identifying antibodies to the organism. -Molecular Methods This techniques Identify organisms by detecting their DNA or RNA by specific technologies. Conventional technologies for identifying microorganisms usually involve good sampling techniques to assure accurate laboratory diagnosis. Conventional Methods for Diagnosis of Infectious Diseases Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world’s biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. Causes of Infectious Diseases: The microbial causes of human diseases are classified into theses groups: 1- Bacteria 2- Viruses 3- Fungi 4- Protoza 5- Chlamydiae 6- Rickettsiae 7- Mycoplasmas. Infection may be endogenous or exogenous. 1- Endogenous infections: the microorganism (usually a bacterium) is a component of the patient's endogenous flora. Endogenous infections can occur when the microorganism is aspirated from the upper to the lower respiratory tract or when it penetrates the skin or mucosal barrier as a result of trauma or surgery. 2- Exogenous infections: the microorganism is acquired from the environment (e.g., from soil or water) or from another person or an animal. The ability to control such microbial infections is largely dependent on the ability to detect these etiological agents in the clinical microbiology laboratory (Millar et al., 2003) Diagnoses of Infectious Diseases Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods: - Conventional Methods Laboratory tests may identify organisms either directly microscopically by stains, growing organisms by cultural techniques and/or detecting their antigens or indirectly by identifying antibodies to the organism. -Molecular Methods This techniques Identify organisms by detecting their DNA or RNA by specific technologies. Conventional technologies for identifying microorganisms usually involve good sampling techniques to assure accurate laboratory diagnosis. Figure (1): Microbiology laboratory techniques for diagnosis of infectious diseases. A-Proper Sampling in Clinical Microbiology Laboratory Specimens selected for microbiologic examination should reflect the disease process and collected in sufficient quantity to allow complete microbiologic examination. The number of microorganisms per milliliter of a body fluid or per gram of tissue is highly variable, ranging from less than 1 to 108 or 1010 colony-forming units (CFU). Collection of good quality specimens depends on: -The optimal time of specimen collection. -The correct type of specimen -Well collected specimens with minimum contamination from normal flora of the patient or the person collecting the specimen. -Adequate amounts of each specimens and appropriate number of specimens -Clearly labeled safe specimens Optimal Time of Collection of Specimens The proper time of sampling in clinical microbiology play a crucial role in proper laboratory diagnosis. There are several examples for such proper time. If possible, specimens should be collected before the administration of antibiotics. Above all, close communication between the clinician and the microbiologist is essential to ensure that appropriate specimens are selected and collected and that they are appropriate -Blood cultures and blood films for malarial parasites are best collected just as the patient’s temperature starts to rise, however, when infective endocarditis is suspected, three blood culture sets collected with 24 hour irrespective of patient, s temperature. - Specimens for virus isolation are most likely to give positive results when collected during the most acute stages of the disease -Serology is satisfactory when four fold or greater rising antibody titer is demonstrated in pained sera. The 1st serum sample as early as possible in the disease course. Second in the convalescent stage. Correct Types of Specimens Examples: Bacterial meningitis-------blood cultures, CSF culture Suspected gonorrhea -------cervical, urethral and rectal swab should be collected rather than high regional swabs. Well Collected Specimens with Minimum Contamination from the Normal Flora The good quality of microbiological sample is a need for accurate laboratory diagnosis. The main problem is mixing of samples with normal flora normally resident beside infected tissue. Skin and mucous membranes have a large and diverse endogenou flora; so every effort must be made to minimize specimen contamination during collection. Contamination may be avoided by various means: - The skin can be disinfected before aspirating or incising a lesion. - The contaminated area may be bypassed altogether. Examples of such approaches are trans tracheal puncture with aspiration of lower respiratory secretions or supra pubic bladder puncture with aspiration of urine. It is often impossible to collect an uncontaminated specimen, and decontamination procedures, cultures on selective media, or quantitative cultures must be used. Specimens collected by invasive techniques, particularly those obtained intra operatively, require special attention. Enough tissue must be obtained for both histopathologic and microbiologic examination. Adequate Amounts of Appropriate Number of Specimens The volume of blood for culture from adult -5-10 ml per bottle and in children and neonates 1-5ml per bottle. -Collection of early morning sputum specimens, and collection of adequate amount of early morning urine specimen for 3 successive days is required for the isolation of Mycobacterium tuberculosis (TB). -Patients with diarrhea ---at least 2 specimens of stool are collected for culture of Salmonellae spp. or Shigella spp. -Serological investigations usually require paired sera. Clearly Labeled and Safe Specimens Specimens for microbiological investigations should be placed in leak – proof containers, and each container should be enclosed in plastic bag. The hazards to staff handling leaking container s include acquiring enteric infection from feces, TB from sputum of an open case of pulmonary TB and viruses s such as HCV, HBV, HIV, from leaking blood. B-Transport of specimens to the laboratory The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations. Many pathogenic organisms don’t survive for long in clinical specimens kept at room temperature. 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 is 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 4hours. 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. Figure (2) Laboratory procedures used in confirming a clinical diagnosis of infectious disease with a bacterial etiology. C- Conventional laboratory Methods for Diagnosis of Infectious Diseases 1-Direct method: They are Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture) I- Microscopy Microscopy can be done quickly, but accuracy depends on the experience of the microscopist and quality of equipment. Regulations often limit physicians' use of microscopy for diagnostic purposes outside a certified laboratory (Siqueira, J. Fet al., 2005). Most specimens are treated with stains that color pathogens, causing them to stand out from the background, although wet mounts of unstained samples can be used to detect fungi, parasites (including helminth eggs and larvae), vaginal clue cells, motile organisms (eg, Trichomonas), and syphilis (via darkfield microscopy). Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms (Fredricks and Relman, 1999). The clinician orders a stain based on the likely pathogens, but no stain is 100% specific. Most samples are treated with Gram stain and, if mycobacteria are suspected, an acid-fast stain. However, some pathogens are not easily visible using these stains; if these pathogens are suspected, different stains or other identification methods are required. Because microscopic detection usually requires a microbe concentration of about 1 × 105/mL, most body fluid specimens (eg, CSF) are concentrated (eg, by centrifugation) before examination. Types of Commonly Used Stains. Gram stain: The Gram stain classifies bacteria according to whether they retain crystal violet stain (Gram-positive—blue) or not (Gram-negative—red) and highlights cell morphology (eg, bacilli, cocci) and cell arrangement (eg, clumps, chains, diploids). Such characteristics can direct antibiotic therapy pending definitive identification. To do a Gram stain, technicians heat-fix specimen material to a slide and stain it by sequential exposure to Gram's crystal violet, iodine, decolorizer, and counterstain (typically safranin). Acid-fast and moderate (modified) acid-fast stains: These stains are used to identify acid-fast organisms (Mycobacterium sp) and moderately acid-fast organisms (primarily Nocardia sp). These stains are also useful for staining Rhodococcus and related genera, as well as oocysts of some parasites (eg, Cryptosporidium). Although detection of mycobacteria in sputum requires only about 5, 000 to 10, 000 organisms/mL, mycobacteria are often present in lower levels, so sensitivity is limited. Usually, several mL of sputum are decontaminated with Na hydroxide and concentrated by centrifugation for acid-fast staining. Specificity is better, although some moderately acid-fast organisms are difficult to distinguish from mycobacteria. Fluorescent stains: These stains allow detection at lower concentrations (1 × 104 cells/mL). Examples are acridine orange (bacteria and fungi), auramine-rhodamine and auramine O (mycobacteria), and calcofluor white (fungi, especially dermatophytes). Coupling a fluorescent dye to an antibody directed at a pathogen (direct or indirect immunofluorescence) should theoretically increase sensitivity and specificity. However, these tests are difficult to read and interpret, and few (eg, Pneumocystis and Legionella direct fluorescent antibody tests) are commercially available and commonly used. India ink (colloidal carbon) stain: This stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, CSF sediment). The background field, rather than the organism itself, is stained, which makes any capsule around the organism visible as a halo. In CSF, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated. Wright's stain and Giemsa stain: These stains are used for detection of parasites in blood, Histoplasma capsulatum in phagocytes and tissue cells, intracellular inclusions formed by viruses and chlamydia, trophozoites of Pneumocystis jiroveci, and some intracellular bacteria. Trichrome stain (Gomori-Wheatley stain) and iron hematoxylin stain: These stains are used to detect intestinal protozoa. The Gomori-Wheatley stain is used to detect microsporidia. It may miss helminth eggs and larvae and does not reliably identify Cryptosporidium. Fungi and human cells take up the stain. The iron hematoxylin stain differentially stains cells, cell inclusions, and nuclei. Helminth eggs may stain too dark to permit identification. Disadvantages of Microscopic methods: (a) Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species. (b) Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator. (c) Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible. (d) Limited specificity is because our inability to speciate micro-organisms based on their morphology and staining patterns. II-Culture Methods Culture is microbial growth on or in a nutritional solid or liquid medium; increased numbers of organisms simplify identification. Culture also facilitates testing of antimicrobial susceptibility (Relman DA., 2002) Communication with the laboratory is essential. Although most specimens are placed on general purpose media (e g, blood or chocolate agar), some pathogens require inclusion of specific nutrients and inhibitors or other special conditions (Wade W., 2002) For more than a century, cultivation using artificial growth media has been the standard diagnostic test in infectious diseases. The microbiota associated with different sites in the human body has been extensively and frequently defined by studies using cultivation approaches. The success in cultivation of important pathogenic bacteria probably led microbiologists to feel satisfied with and optimistic about their results and to recognize that there is no death of known pathogens (Relman, 1992 and Wade W, 2002). But should we be so complacent with what we know about human pathogens? Making micro-organisms grow under laboratory conditions presupposes some knowledge of their growth requirements. Nevertheless, very little is known about the specific growth factors that are utilized by innumerous micro-organisms to survive in virtually all habitats, including within the human body (Wade, 2002). A huge proportion of the microbial species in nature are difficult to be tamed in the laboratory. Certain bacteria are fastidious or even impossible to cultivate. Some well-known human pathogens, such as Mycobacterium leprae and Treponema pallidum continue to defy scientists regarding their cultivation under laboratory conditions (Fredricks and Relman, 1999) need more read Recent advances in diagnosis of infectious diseases http://www.amazon.co.uk/Advances-Diagnosis-Infectious-Diseases-Laboratory/dp/3848445751

Meningitis

Meningitis Meningitis and encephalitis are potentially life threatening infections especially in children. Meningitis is defined as an inflammation of the meninges, the tough layer of tissue that surrounds the brain and the spinal cord. Aseptic meningitis (AM) is an inflammation of the meninges which is caused mainly by nonbacterial organisms. AM denotes a clinical syndrome characterized by fever , neck stiffness and may be convulsions with a predominance of lymphocytes in the CSF with negative bacterial culture of the CSF. Viral meningitis occurs as an uncommon complication of systemic viral infection that occurs most frequently in infants and children. Morbidity and mortality depend on the infectious agent, age of the child, general health and prompt diagnosis and treatment. Many etiological agents can cause AM .Viruses are the most frequent causes as Enterovirus,Poliovirus , Coxsackievirus ,Echoviruses,Herpes simplex virus (HSV) ,Varicella-zoster virus( VZV) ,Cytomegalovirus (CMV) , Epstein-Barr virus (EBV). Bacterial infections such as tuberculosis , mycoplasma and leptospira can also cause AM. Non infectious causes may include postvaccination with MMR ,rabies and poliomylities vaccines , drugs as nonsteroidal anti-inflammatory drugs (NSAIDs) and antibiotics as amoxicillin, trimethoprim–sulfamethoxazole. Also fungi as Candida , Cryptococcus and parasites as Toxoplasma gondii , Trichinosis , Neurocysticercosis and Naeglari are infrequent causes of AM. Enteroviruses (EVs) especially non polio EVs (NPEV) including echovirus and coxsackie A and B viruses are the most common etiologic agents of AM . EVs meningitis can mimic bacterial meningitis. So, it is important to distinguish AM, meningoen¬cephalitis from bacterial meningitis which demands prompt therapeutic approach and to avoid unnecessary hospitalization and antibacterial treatment in cases AM. Laboratory diagnosis of AM depends on nonspecific tests as macroscopical examination , cell count both total and differential leucocytic count,direct Gram stained ,cell culture and biochemical tests as glucose , protein ,CSF lactate , C-Reactive protein and CSF adenosine deaminase (ADA). The specific tests are virus isolation, virus antigen detection, virus nucleic acid detection and virus antibody detection (serology). Virus isolation is the current method of choice for the diagnosis but it takes several days to be conclusive. However, attempts to isolate virus from CSF are frequently unsuccessful because of the low viral titer in clinical specimens and several types of viruses do not grow well or not at all in tissue culture . Virus antigen detection is more rapid but still manually intensive and relatively insensitive. Virus serology is an indirect approach with many limitations. It is used to assess immune status and to detect the viruses which cannot be cultivated in cell culture.It includes indirect fluorescent antibody testing and enzyme-linked immunosorbent assays (ELISA) which detect antibodies against viruses. The performance of viral serology is useful in the diagnosis of recent, past or chronic viral infections. However , ELISA is not conclusive in the diagnosis of acute EV or HSV meningitis as IgM antibodies may persist for months and it is not unique to the primary phase. There is a great clinical need to develop rapid and sensitive virus diagnostic techniques. Molecular diagnosis may have a significant benefit . PCR is an in vitro method for specific or target cDNA or RNA amplification. The target DNA-or RNA is derived from clinical specimens or a microbial culture. The major advantages of PCR are its rapidity, sensitivity and robustness. But the major disadvantages of PCR are short size,limiting amounts of product and infidelity of DNA replication . The specificity of PCR is based on the sequence of the two primers. This means that any segment of genomic DNA or RNA is a potential target for PCR diagnostic assay. An ideal target sequence should be found in all strains of the virus of interest but not found in any other viruses. Conclusions : * Meningitis is the most common infectious CNS syndrome which is a life – threatening condition especially in children . * Aseptic meningitis is caused mainly by viruses as Enterovirus , Polioviruses , Coxsackievirus ,Herpes simplex virus ,Varicella-zoster virus,Cytomegalovirus.Other causes may include bacterial as tuberculosis , mycoplasma and leptospira or followed MMR ,rabies and poliomyelitis vaccines.Also , drugs as nonsteroidal anti-inflammatory , trimethoprim or fungi as Candida , Cryptococcus and parasites as Toxoplasma gondii , Trichinosis can cause aseptic meningitis. * Enteroviruses are the most common causes of viral meningitis causing appreciable morbidity. * Molecular biological methods as PCR has been developed. It becomes the technique of choice for detecting viral or other pathogen genome. Laboratory Diagnosis of Meningitis Made Ridiculously easy [Kindle Edition] http://www.amazon.co.uk/Laboratory-Diagnosis-Meningitis-Ridiculously-ebook/dp/B007GIXXR6

The Use of the Clinical Microbiology Laboratory (General Principles )

* Collection of Clinically Relevant Specimens:


The best use of the laboratory involves sending only relevant specimens so that work can be done on a reasonable no. of specimens especially when economic situation of the country is greatly limited

Examples of unnecessary investigations include routine microscopy and culture of urine specimens from all non catheterized adult patients in hospital without symptoms suggestive of urinary tract infection.

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

Proper Sampling in Clinical Microbiology Laboratory

Collection of good quality specimens:

Depends on

1- The optimal time of specimen collection.

2- The correct type of specimen

3- Well collected specimens with minimum contamination from normal flora of the patient or the person collecting the specimen.

4- Adequate amounts of each specimens and appropriate no. of specimens

5- Clearly labeled safe specimens

1- Optimal time of collection of collection of specimens:

-specimens for the culture of bacteria collected before the start of antibiotic therapy

-Blood cultures and blood films for malarial parasites are best collected just as the patient’s temp starts to rise, however, when infective endocarditis is suspected, three blood culture sets collected with 24 hour irrespective of patient temp.

- Specimens for virus isolation are most likely to give positive results when collected during the most acute stages of the disease

-Serology is satisfactory when four fold or greater rising antibody titer is demonstrated in pained sera.

The 1st serum sample as early as possible in the disease course. Second in the convalescent stage

2-Correct types of specimens:

Examples:

Bacterial meningitis-------blood cultures CSF culture

Suspected gonorrhea -------cervical, urethral and rectal swab should be collected rather than high regional swabs.

3-Well collected specimens with minimum contamination from the normal flora:

Poor quality specimens include saliva instead of sputum or a salivary – mucoid sputum sample instead of a muco purulent sputum.

-Mid stream urine need careful collection to a void excess contamination by genital flora.

-A throat swab should not touch the buccal mucosa and the tongue depressed by a spatula.

-Vaginal speculum should not be wet with antiseptic solution during collection of high vaginal swab with care not to touch the lower vagina or perineum.

-Strict septic and antiseptic techniques are used for blood and CSF cultures to avoid contamination from skin flora or from the doctor.

4-Adeqate amounts of appropriate number of specimens:

The volume of blood for culture from adult

-5-10 ml per bottle and in children and neonates 1-5ml per bottle.

-Collection of early morning sputum specimens, and collection of adequate amount of early morning urine specimen for 3 successive days is required for the isolation of M.T.B.

-Patients with diarrhea ---at least 2 specimens of faeces is collected for culture of Salmonellae or Shigella.

-Serological investigations usually require paired sera.

5-Clearly labeled and safe specimens:

Specimens for microbiological investigations should be placed in leak – proof containers, and each container should be enclosed in plastic bag.

The hazards to staff handling leaking container s include acquiring enteric infection from feces, T.B. from sputum of an open case of pulm. T.B. and viruses s such as HCV, HBV, HIV, from leaking blood.

Transport of 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.

Basic Laboratory procedures for microbiological diagnosis

Laboratory procedures for microbiological diagnosis Include the following steps:

-Naked eye examination of Specimens

-Microscopy.

-Detection of microbial antigens.

-Isolation of microbes

- Antibiotic sensitivity

-Serology.

-Molecular biology techniques.

-Gas – liquid chromatographic techniques.

-Skin tests.

I. Naked eye examination of specimens:-

This helps to determine whether a specimen is suitable or no.

- A saliva sample instead of an expectorated sputum sample should be discarded.

-Turbid CSF, is an immediate evidence of infection

-A foul smelling pus specimen may suggest presence of anaerabes.

-A rice water stool sample may indicate Vibrio cholera infection

-Anchory sauce sputum sample would suggest invasive Amoebiasis in lungs.

-Sulphar granules in pus would indicate Actinomycosis.

II. Microscopy:

1-Wet preparation for light microscopy in examination of CSF, urine, body fluid for evidence of pus cells, and organisms.

-Vaginal secretion-- Trichomonas and Candida .

-Skin, nail , hair (in KaOH) -evidence of fungus

-Dark ground illumination to look for Spirochates, Treponema pallidum in suspected 1ry or 2ry .

2-Gram stained smear: It may help of saving life.

-Important in rapid diagnosis of bacterial meningitis on exam of CSF deposit.

-Diagnosis of Strepto pneumonia in sputum smear

-Performed in serious septicemia when a blood culture bottle is flagged positive.

- Identification of colonies appearing on culture media

-Gram – stained smear may give positive results al though the subsequent cultures are negative as a result of given antibiotics

-In Vincent's angina: stained smear the only means of diagnosis

3-Acid fast stain of sputum allow rapid diagnosis of open pulmonary tuberculosis demonstrating acid fast bacilli. In other, clinical specimens such as urine, peritoneal fluid, CSF it lacks sensitivity.

4-Immunoflourescent microscopy is important for rapid diagnosis of viral infection e.g. Respiratory syncytial virus (RSV) in infants and children, Herpes simplex, cytomegalovirus (CMV) in urine throat swab, Rabies in brain biopsy specimen, Clamydia trachomatis in conjunctival scrapings. Also, it can be used in serological antibody test e.g Fluorescent Treponema antibody and fluorescent amoebic antibody.

5-Eectron Microscopy:

Mainly used for rapid diagnosis of rota virus or herpes infection, CMV in neonatal urine specimens as the virus voids in urine in large amount.

III- Detection of microbial antigens:

Becomes increasingly important in recent years.

- Immunoelectrophoresis . e.g Pneumococcal polysaccharide antigen may be detected in sputum, serum, urine of patients by immunoelectrophoresis, when patients have already given antibiotics where as conventional sputum blood cultures are negative.

- Hepatitis B surface antigen (HbsAg) is commonly detected using ELISA or latex test

-Cryptococcal antigen in CSF in patients with cryptococcal meningitis with latex.

-Rota virus antigen using enzyme linked immunosorbant assay(ELISA) in feces of diarrheic children infected with rota virus.

-Chlamydia trachomatis antigen using ELISA in conjunctival scrappings in patients with active trachoma.

-Detection of Meningococcae, Hemophilus, Pneumococcae antigen in CSF specimens by latex

-Commercially available monoclonal specific antibodies for detection of antigen in clinical specimens or cultures including various Streptococcal, Staphylococcal species, Neisseria, Candida spp. Chlamydia trachomatis, and Rotariruses, CMV, Herpes, Adeno viruse, RSV, Influenza viruses.

IV- Isolation of microbes

Is the most reliable way in which a diagnosis can be confirmed and for obtaining antimicrobial susceptibility results.

-Isolation of bacteria or fungus from specimens such as Blood, CSF (which are normally sterile) are easy to interpret. While, bacterial or fungal isolation from specimens collected from sites with normal flora are often difficult to interpret.

-Choice of media is important according to type of specimen and suspected organism.

-Virological or chlamydial isolation methods. Need preparation of the cell line required for support growth of suspected infecting virus.

For Chlamydia -----Vero, Maccoy line

For Adeno - Vero , HEP

For CMV- Human diploid fibroblast.

For Influenza------ chick embryo.

V-Antimicrobial susceptibility testing

1-Disc diffusion tests

Limitation of disc diffusion tests:-

-Not applied to slowly growing, Fastidious organisms or anaerobes .

-Mycobacterial and fungus susceptibility testing requires specific techniques

-The reported sensitivity tests results not applied to clinical sites infections, e.g Salmonella typhi to aminoglycosids.

-Not related to the achieved serum levels or body fluid levels of antibiotics.

-Bacteriostatic measures only.

-Can’t be applied to certain antibiotics such as polymyxines.

2-Dilution susceptibility testes:-

Micro minimal inhibitory and minimal bactericidal activity methods.

Methods

-Broth dilution tests

-Agar dilution method.

Application:-

-Serious infection where endpoint concentration is ended

-Disc diffusion yield inter mediate susceptibility

-Life threatening infection due to organisms with unpredictable susceptibility pattern.

-Fastidious or slowly growing organisms.

-Failure of antibiotic therapy

-Serious infections caused by organisms susceptible only to toxic agents

Limitations

-Difficult

It needs the knowledge about the achievable level in serum or body filmed

3- Automated method

4-Antimicrobial concentration gradient methods

-A serial antibiotic dilations are incorporated into the agar.

-E test

VI-Serology

Ten days or longer has to pass before arising antibody demonotrated

in chronic infections. e.g , Brucellosis serology is often available

-IgM antibody may indicates recent infection (e.g Rubella)

-ELISA technique widely used nowadays for detection of ab

Immune status of the patients immunization should be taken in considerations.

VII-Molecular biology techniques:

Increasingly important for rapid diagnosis of infections for epidemiological investigations and for monitoring antimicrobial therapy

Also, important for research on the pathogenesis of infection, the developmental new vaccines and immune therapeutic agents.

VIII Gas Liquid chromatographic techniques:

Become increasingly useful for the rapid detection of anaerobic infections

Specimens of pus from abdominal, gynecological or brain abscesses may be shawn to have multiple volatile fatty acids present which indicate anaerobic infection with a few hours of collection of the specimen of pus and this may affect decisions about the chemotherapy of infection.

IX- Skin test:

Of limited value for diagnosis of infection ex:

-Mantaux skin test for tuberculosis.

-Histoplasmin test for Histoplasma infection.

-Casoni test

-Schick test