1. DIAGNOSING INFECTIOUS DISEASES Dr. Mohammad Shakeeb,MD Specialist in clinical pathology/microbiology and immunology

2. INTRODUCTION The proper diagnosis of an infectious disease requires taking a complete patient history physical examination of the patient Carefully evaluating the patient’s signs and symptoms implementing the proper selection, collection, transport,and processing of appropriate clinical specimens

3. CLINICAL SPECIMENS

4.  Role of Healthcare Professionals in the Submission of Clinical Specimens The doctor, nurse, medical technologist, or other qualified healthcare professional must select the appropriate specimen, collect it properly, and then properly transport it to the CML for processing. Laboratory findings must then be conveyed to the attending clinician as quickly as possible to facilitate the prompt diagnosis and treatment of the infectious disease.

5. Healthcare personnel who collect clinical specimens must strictly adhere to the safety policies known as Standard Precautions. All specimens should be collected or transferred into a leak- proof primary container with a secure closure.

6.  Importance of High-Quality Clinical Specimens High-quality clinical specimens are required to achieve accurate, clinically relevant laboratory results. The three components of specimen quality are: o Proper specimen selection o proper specimen collection o Proper transport of the specimen to the laboratory The laboratory must provide written guidelines regarding specimen selection, collection, and transport in the form of a manual.

7.  Proper Selection, Collection, and Transport of Clinical Specimens Appropriate type of specimen for diagnosis of the suspected infectious disease. Specimens must be collected in a manner that will eliminate or minimize contamination of the specimen with indigenous microflora. Specimens should be obtained before antimicrobial therapy has begun. The acute stage of the disease is the appropriate time to collect most specimens.

8. If the patient is to collect the specimen, such as sputum or urine, the patient must be given clear and detailed collection instructions. A sufficient quantity of the specimen must be obtained to provide enough material for all required diagnostic tests. All specimens should be placed or collected into a sterile container. Specimens should be protected from heat and cold and promptly delivered to the laboratory.

9. Specimens must be placed in a sealed plastic bag for immediate and careful transport to the laboratory. The specimen container must be properly labeled and accompanied by an appropriate laboratory test requisition containing adequate instructions. Labels should contain : The patient’s name. Unique hospital identification number. Hospital room number. Requesting clinician’s name. Culture site. Date and time of collection.

10. Laboratory test requisitions should contain: The patient’s name, age, sex. Unique hospital identification number. Name of the requesting clinician. Specific information about the type of specimen. The site from which it was collected. Date and time of collection. Initials of the person who collected the specimen. Information about any antimicrobial agent(s) that the patient is receiving.

11.  Contamination of Clinical Specimens with Indigenous Microflora. when present in specimens, these organisms might merely be contaminants, but it is also possible that they are causing an infection. (Opportunistic pathogens).

12.  Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases.  Blood  Urine  Cerebrospinal Fluid  Sputum  Throat Swabs  Wound Specimens  Fecal Specimens

13.  Blood Blood is usually sterile. The presence of bacteria in the bloodstream (bacteremia) may indicate a disease. temporary or transient bacteremias may occur after oral surgery, tooth extraction, or even aggressive tooth brushing that causes bleeding. Bacteremia may occur during certain stages of many infectious diseases.

14. Septicemia is a serious disease characterized by chills, fever, prostration, and the presence of bacteria or their toxins in the bloodstream. The most severe types of septicemia are those caused by Gram-negative bacilli, owing to the endotoxin that is released from their cell walls. Endotoxin can induce fever and septic shock, which can be fatal. To diagnose either bacteremia or septicemia, it is recommended that at least three blood cultures be collected during a 24-hour period.

15.  The person drawing the blood must wear sterile gloves, and gloves must be changed between patients.  Blood for culture is usually obtained from a vein located at the antecubital fossa.  After locating a suitable vein, the skin at the site is disinfected with 70% isopropyl alcohol and then with an iodophor.  When disinfecting the site, a concentric swabbing motion is used.  The iodophor is then allowed to dry.  A tourniquet is applied and the appropriate amount of blood is withdrawn.

17.  It is important not to touch the site after it has been disinfected.  there are many different types of blood culture systems currently available.  The rubber tops of blood culture bottles must be disinfected prior to insertion of the needle.  Then an appropriate volume of blood is injected.  The blood culture bottle(s) should be transported promptly to the laboratory for incubation at 37°C.

19.  Urine Urine is ordinarily sterile while it is in the urinary bladder. during urination, it becomes contaminated by indigenous microflora of the distal urethra. Contamination can be reduced by collecting a clean-catch, midstream urine. “Clean-catch”: area around the external opening of the urethra is cleansed by washing with soap and rinsing with water before urinating. removes the indigenous microflora.

20. “Midstream”: refers to the fact that the initial portion of the urine stream is directed into a toilet or bedpan, and then the urine stream is directed into a sterile container. the microorganisms that live in the distal urethra are flushed out of the urethra by the initial portion of the urine stream, into the toilet or bedpan, rather than into the specimen container. the clinician may prefer to collect a catheterized specimen or use the suprapubic needle aspiration technique to obtain a sterile sample of urine.

21. all urine specimens must be processed within 30 minutes of collection, or refrigerated at 4°C until they can be analyzed. Refrigerated urine specimens should be cultured within 24 hours. Failure to refrigerate a urine specimen will cause an inflated colony count------incorrect diagnosis of UTI.

22. Complete urine culture consists of a colony count, isolation and identification of the pathogen, and antimicrobial susceptibility testing. A calibrated loop is used to perform the colony count. contains a precise volume of urine. two types: calibrated to contain 0.01 ml. calibrated to contain 0.001 ml.  The calibrated loop is dipped into the CCMS urine specimen.  volume of urine within the calibrated loop is inoculated over the entire surface of a blood agar plate, which is then incubated overnight at 37°C

23. After incubation, the colonies are counted and this number is then multiplied by the dilution factor (either 100 or 1,000) to obtain the number of colony-forming units (CFU) per milliliter of urine. The dilution factor is 100 if a 0.01-mL calibrated loop was used, or 1,000 if a 0.001-mL calibrated loop was used. A CFU count that is 100,000 (1 × 10*5) CFU/mL or higher is indicative of a UTI. A CFU count that is 100,000 (1 × 10*5) CFU/mL or higher is indicative of a UTI.

24. Cerebrospinal Fluid Cerebrospinal Fluid Meningitis is inflammation or infection of the membranes (meninges) that surround the brain and spinal column. Encephalitis is inflammation or infection of the brain. Meningoencephalitis is inflammation or infection of both the brain and the meninges. To diagnose these diseases, CSF must be collected into a sterile tube by a lumbar puncture (spinal tap) under surgically aseptic conditions. difficult procedure is performed by a physician.

25. CSF specimens must be rushed to the laboratory and must not be refrigerated. Refrigeration might kill any fragile pathogens present in the specimen. Emergency specimen. workup of the specimen will be initiated immediately. Gram stain of the spinal fluid sediment will be reported by telephone to the clinician immediately. enable clinicians to make diagnoses and initiate therapy, and often save patients’ lives.

26.  Sputum Sputum is pus that accumulates deep within the lungs of a patient with pneumonia, tuberculosis, or other lower respiratory infection. Many of the sputum specimens that are submitted to the CML are actually saliva. If tuberculosis is suspected, extreme care in collecting and handling the specimen should be exercised because one could easily be infected with the pathogens. Sputum specimens may be refrigerated for several hours without loss of the pathogens.

27.  Throat Swabs Routine throat swabs are collected to determine whether a patient has strep throat (Streptococcus pyogenes pharyngitis). If a clinician suspects a pathogen other than S. pyogenes to be causing a patient’s pharyngitis, that information must be included on the laboratory test requisition.

28.  Wound Specimens Wound specimen should be an aspirate (using a small needle and syringe) rather than a swab specimen. swab are frequently contaminated with indigenous skin microflora and often dry out The laboratory test requisition that accompanies a wound specimen must indicate the type of wound and its anatomical location.

29.  Fecal Specimens Ideally, fecal specimens (stool specimens) should be collected at the laboratory and processed immediately. Alternatively, the specimen may be placed in a container with a preservative that maintains a pH of 7.0. In gastrointestinal infections, the pathogens frequently overwhelm the indigenous intestinal microflora, so that they are the predominant organisms seen in smears and cultures. Enteropathogenic Escherichia coli, Salmonella spp., Shigella spp., Clostridium perfringens, C. difficile, Vibrio cholerae, Campylobacter spp.

30. THE PATHOLOGY DEPARTMENT (“THE LAB”)

32. THE CLINICAL MICROBIOLOGY LABORATORY

33. The four major responsibilities of the CML are: Processing clinical specimens. Isolate pathogens. Identifying pathogens Perform antimicrobial susceptibility testing when appropriate to do so.

35. In general, the processing of clinical specimens in the CML includes : Examining the specimen macroscopically. Examining the specimen microscopically. Inoculating the specimen to appropriate culture media. A less frequent responsibility of the CML is to process environmental samples whenever there is an outbreak or epidemic within the hospital.

36. Environmental samples include those collected from appropriate hospital sites (floors, sink drains, showerheads, respiratory therapy equipment.), and employees (e.g., nasal swabs, material from open wounds). To isolate bacteria and fungi from clinical specimens, specimens are inoculated into liquid culture media or onto solid culture media.

37.  Bacteriology Section CML professionals gather “clues” (phenotypic characteristics) about a pathogen until they have sufficient information to identify (speciate) it.

38. Gram reaction. Cell shape. Morphologic arrangement of cells. Growth or no growth on various types of plated media. Colony morphology. Presence or absence of a capsule Motility Number and location of flagella Ability to sporulate Location of spores Presence or absence of various enzymes (e.g., catalase, coagulase, oxidase, urease) Ability to catabolize various carbohydrates and amino acids Ability to reduce nitrate Ability to produce indole from tryptophan Atmospheric requirements Type of hemolysis produced

39.  Mycology Section Three types are specimens are much more commonly submitted to the Mycology Section than to the Bacteriology Section: hair clippings. nail clippings. skin scrapings. A potassium hydroxide preparation (KOH prep) is performed on hair clippings, nail clippings, and skin scrapings.

40. The KOH acts as a clearing agent by dissolving keratin in the specimens. This enables the technologist to see into the specimens when they are examined microscopically. determine whether any fungal elements (e.g., yeasts or hyphae) are present in the specimen. Specimens will also be inoculated onto Sabouraud dextrose agar, a selective medium for fungi. When isolated from clinical specimens, yeasts are identified using various biochemical tests, primarily based on their ability to catabolize various carbohydrates.

41. When isolated from clinical specimens, moulds are identified using a combination of rate of growth and macroscopic and microscopic observations. Susceptibility testing of fungi is not currently performed in most CMLs.

42.  Parasitology Section Parasitic infections are diagnosed by observing and recognizing various parasite life cycle stages (e.g., trophozoites and cysts of protozoa; microfilariae, eggs, and larvae of helminths) in clinical specimens.

43.  Virology Section Many viral diseases are diagnosed using immunodiagnostic procedures. Other techniques used to identify viral pathogens are:  Observation of intracytoplasmic or intranuclear viral inclusion bodies in specimens by cytological or histological examination.  Observation of viruses in specimens using electron microscopy.  Molecular techniques  Virus isolation by use of cell cultures.

44.  Mycobacteriology Section Various types of specimens (primarily sputum specimens) are processed. Acid-fast staining is performed. Mycobacteria are isolated and identified. Susceptibility testing is performed. Mycobacterium spp. are identified using a combination of growth characteristics and various biochemical tests.