13 DIAGNOSING INFECTIOUS DISEASES: 13 DIAGNOSING INFECTIOUS DISEASES The Proper Diagnosis of an Infectious Diseases Requires the following:: The Proper Diagnosis of an Infectious Diseases Requires the following: Taking complete patient history Conducting a thorough 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 . Clinical Specimens: Clinical Specimens The various types of specimens (e.g., blood, urine, feces, cerebrospinal fluid) that are collected from patients and used to diagnose or follow the progress of infectious diseases are referred to as clinical specimens The most common types of clinical specimens that are sent that are sent to the hospital ‘s microbiology laboratory (hereafter referred to as the Clinical Microbiology Laboratory or CML .) Types of Clinical Specimens Submitted to the Clinical Microbiology Laboratory: Types of Clinical Specimens Submitted to the Clinical Microbiology Laboratory TYPES OF SPECIMEN TYPE(S) OF INFECTIOUS DISEASE THAT THE SPECIMEN IS USED TO DIAGNOSE TYPES OF SPECIMEN TYPE(S) OF INFECTIOUS DISEASE THAT THE SPECIMEN IS USED TO DIAGNOSE BLOOD B, F, P, V HAIR CLIPPINGS F BONE MARROW B NAIL (FINGERNAIL AND TOENAIL CLIPPINGS) F BRONCHIAL AND BRONCHOALVEOLAR WASHES V NASAL SWABS B CEREBROSPINAL FLUID (CSF) B, F, P, V PUS FROM A WOUND OR ABSCESS B CERVICAL AND VAGINAL SWABS B “SNOTCH TAPE PREP” P CONJUBCTIVAL SWAB OR SCRAPING B,V SKIN SCRAPINGS F FECES AND RECTAL SWABS B, P, V SKIN SNIP P Slide 5: TYPES OF SPECIMEN TYPE(S) OF INFECTIOUS DISEASE THAT THE SPECIMEN IS USED TO DIAGNOSE SPUTUM B, F, P SYNOVIAL (JOINT) FLUID B THROAT SWABS B, V TISSUE (BIOPSY AND AUTOPSY) SPECIMENS B, F, P, V URETHRAL DISCHARGE MATERIAL B URINE B, P, V UROGENITAL SECRETIONS (E.G., VAGINAL DISCHARGE MATERIAL, PROSTATIC SECRETIONS) B, P VESICLE FLUID OR SCRAPING V Role of Healthcare Professionals in the Submission of Clinical Specimens: Role of Healthcare Professionals in the Submission of Clinical Specimens A close working relationship among the members of the healthcare team is essential for the proper diagnosis of the infectious diseases. When an attending physician suspects that a patient has a particular infectious disease, appropriate clinical specimen must be obtained and certain diagnostic tests may be requested. The doctor, nurse, medical technologist, or other qualified healthcare professionals must select an appropriate specimen, collect it properly, and then transport it to the laboratory where it is processed. Slide 7: Laboratory findings must then be conveyed to the A.P. as quickly as possible to facilitate the prompt diagnosis and treatment of the infectious disease . Healthcare professionals who collect and transport clinical specimens should exercise extreme caution during the collection and transport of clinical specimens to avoid sticking themselves with needles, cutting themselves with other types of sharps or coming in contact with any type of specimen. Healthcare personnel who collect clinical specimens must strictly adhere to the safety policies known as standard precautions. Slide 8: According to the Clinical and Laboratory Standards Institute (CLSI), “ All specimen should be collected or transferred into a leak proof of primary container with a secure closure. Care should be taken by the person collecting the specimen not to contaminate the outside of primary container…” Within the institution, the primary container should be placed into a second container, which will contain the specimen if the primary container breaks or leaks in transit to laboratory.” Within the laboratory , all specimens are handled carefully, following standard precautions and ultimately disposed of as infectious waste. Importance of High-Quality Clinical Specimen: Importance of High-Quality Clinical Specimen High-quality clinical specimens are required to achieve accurate, clinically relevant laboratory results (e.g., results that provide information about the patient’s infectious disease.) It has often stated that the quality of the laboratory work performed in the CML can be only as good as the quality of specimens that are received. It is impossible for the CML to obtain and report high-quality test results if the laboratory receives poor-quality specimens or the wrong types of specimens. Slide 10: The laboratory must provide written guidelines regarding specimen selection, collection and transport in the form of a book (although the name of the book varies from one institution to the next, it is referred to in this book as the “Laboratory Policies and Procedures Manual” Copies of the Lab P and P Manual must be available to every ward, floor, clinic and department. Often it is accessible through the hospital’s computer system. However, the person who collects the specimen is ultimately responsible for its quality. The Three Components of Specimen Quality are the following: : The Three Components of Specimen Quality are the following : Proper specimen selection (e.g., the correct type of specimen must be submitted) Proper specimen collection Proper transport of the specimen to the laboratory. When Clinical Specimens are Improperly Collected and Handled:: When Clinical Specimens are Improperly Collected and Handled: The etiologic (causative) agent may not be found or may be destroyed Overgrowth by indigenous microflora may mask the pathogen Contaminants may interfere with the identification of the pathogens and the diagnosis of the infectious disease. Proper Selection, Collection and Transport of Clinical Specimen: Proper Selection, Collection and Transport of Clinical Specimen When collecting clinical specimen for microbiology, these general precautions should be taken: The specimen must be properly selected. The specimen must be properly and carefully collected. The material should be collected from the site where the suspected pathogen is most likely to be found and where the least contamination is likely to occur. Slide 14: Whenever possible, specimens should be obtained before antimicrobial therapy has begun. If this is not possible, the laboratory should be informed as to which the antimicrobial agent(s) the patient is receiving. The acute stage of the disease (when the patient is experiencing the symptoms of the disease) is the most appropriate time to collect most specimens. Specimen collection should be performed with care and tact to avoid harming the patient, causing discomfort, or causing undue embarrassment. Slide 15: A sufficient quality 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 to prevent contamination of the specimen by indigenous microflora and airborne microbes. Specimens should be protected from heat and cold and promptly delivered to the laboratory so that the results of the analysis will validly represent the number and types of organisms present at the time of collection. Slide 16: Hazardous specimens must be handled with even greater to avoid contamination of the courier, patients and healthcare professionals. Whenever possible, sterile, disposable specimen container should be used. The specimen container must be properly labeled and accompanied by an appropriate request slip containing adequate instructions. Specimens should be collected and delivered to the laboratory as early in the day as possible to give the technologists sufficient time to process the material especially when the hospital or clinic does not have 24 hours laboratory service. Types of Clinical Specimen Usually Required to Diagnose Infectious Diseases : Types of Clinical Specimen Usually Required to Diagnose Infectious Diseases Specific techniques for the collection and transport of clinical specimens vary from institution to institution and are contained in the institution’s Lab P and P Manual. BLOOD. It is usually sterile. The presence of bacteria in the bloodstream (bacteremia) may indicate a disease although temporary or transient bacteremia may occur after oral surgery, tooth extraction or even aggressive tooth brushing. Bacteremia may occur during certain stages of many infectious diseases. Slide 18: These diseases include bacterial meningitis, typhoid fever and other salmonella infections, pneumococcal pneumonia, urinary infections , endocarditis, brucellosis, tularemia, plague, anthrax, syphilis and wound infections caused by B-hemolytic streptococci, staphylococci and other invasive bacteria . 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, 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. Slide 19: To prevent contamination of the blood specimen with indigenous skin flora, extreme care must be taken to use sterile technique when collecting blood for culture. The person drawing the blood must wear sterile gloves and gloves must be changed between patients. After locating a suitable vein, disinfect the skin with 70 % isopropyl alcohol and then with an iodophor . Slide 20: When disinfecting the site, use a concentric swabbing motion, starting at the point at which you intend to insert the needle a nd working outward from that point. Allow the iodophor to dry. Apply a tourniquet and then withdraw the appropriate amount of blood. Do not touch the site after it has been disinfected. Slide 21: Traditionally, the blood has been injected into two blood culture bottles (one aerobic bottle and anaerobic bottle), but there are many different types of blood culture systems currently available. Always disinfect the rubber tops of blood culture bottles before insertion of the needle. Inject the volume of blood specified for the type of blood culture being used. After venipuncture, remove the iodophor from the skin with alcohol. The blood culture bottle(s) should be tranporeted promptly to the laboratory for incubation at 37 degrees C. Blood culture bottles should not be refrigerated. Urine: Urine Urine is ordinarily sterile while it is in the urinary bladder. However during urination, it becomes contaminated by indigenous microflora of the distal urethra (the portion of the urethra farthest from the bladder.) Contamination can be reduced by collecting a clean-catch, midstream urine (CCMS urine). “Clean-catch” refers to the fact that the area around the external opening of the urethra is cleansed by washing with soap and rinsing with water before urinating. Slide 23: “Midstream” refers to the fact that the initial portion of the urine stream is directed into a sterile container. Thus 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. In some circumstances, the physician may prefer to collect a catheterized specimen or the use of suprapubic needle aspiration technique to obtain a sterile sample of urine. Slide 24: In the latter technique, a needle is inserted through the urinary the abdominal wall into the urinary bladder and a syringe is used to withdraw urine from the bladder. To prevent continued bacterial growth, all urine specimens must be processed within 30 minutes of collection or refrigerated at 4 degrees 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 which could lead to an incorrect diagnosis of a urinary tract infection (UTI). Three Parts of Urine Culture: Three Parts of Urine Culture A colony count Isolation and identification of the pathogen Antimicrobial susceptibility testing Slide 26: The colony count is the way of estimating the number of viable bacteria that are present in the urine specimen. A calibrated loop is used to perform a colony count. A calibrated loop is a bacteriologic loop that has been manufactured so that it contains a precise volume of urine. There are two types of calibrated loops: those calibrated to contain 0.01 mL of fluid and those calibrated to contain 0.001 mL of fluid. Slide 27: The calibrated loop is dipped into the CCMS urine specimen. Then the volume of the urine within the calibrated loop is inoculated over the entire surface of a blood agar plate, which is then incubated overnight at 37 degrees C. 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. Slide 28: The mere presence of bacteria in the urine ( bacteriuria ) is not significant, as urine always contaminated with the bacteria during urination (voiding). However the presence of two or more bacteria per x 1,000 microscopic field of a Gram-stained urine smear is indicative of a UTI with 100, 000 or more CFU per milliliter. Cerebrospinal Fluid: Cerebrospinal Fluid Meningitis, encephalitis and meningoencephalitis are rapidly fatal diseases that can be caused by a variety of microbes including bacteria, fungi, protozoa and viruses. Meningitis is the inflammation or infection of the membranes (meninges) that surround the brain and the spinal column. Slide 30: Encephalitis is the inflammation or infection of the brain. Meningoencephalitis is the inflammation or infection of both the brain and the meninges. To diagnose these diseases, cerebrospinal fluid (also referred to as spinal fluid CSF) must be collected into a sterile tube a lumbar puncture (spinal tap) under a surgically aseptic conditions. This procedure is performed by a physician. CSF specimens must be rushed to the laboratory and must not be refrigerated. Slide 31: Because of extremely serious nature of central nervous system (CNS) infections, the CSF will be treated as a STAT (emergency) specimen in the CML and a workup of specimen will be initiated immediately. Information obtained as result of examining a Gram stain of the spinal fluid sediment will be reported by telephone to the physician immediately; this is what is known as preliminary report. Preliminary reports are laboratory reports that are communicated (usually by telephone) to the requesting physician before the availability of the final report. Sputum: Sputum Sputum is the pus that accumulates deep within the lungs of a patient with pneumonia, tuberculosis or other lower respiratory infection. Unfortunately, many of the sputum specimens that are submitted to the CML are actually saliva . This situation can be avoided if someone (most often, a nurse) takes a moment to explain to the patient what is required. Slide 33: If proper mouth hygiene is maintained, the sputum will not be severely contaminated with oral flora. If tuberculosis is suspected, extreme care in collecting and handling the specimen should be exercised because one could easily be infected with pathogens. Usually, sputum specimens may be refrigerated for several hours without loss of pathogens. Slide 34: The physician may wish to obtain a better quality specimen by bronchial aspiration through a bronchoscope or a by a process known as transtracheal aspiration. Needle biopsy of the lungs may be necessary for diagnosis of Pneumocystis jiroveci pneumonia (as in patients with AIDS) and for certain other pathogens. Throat Swabs: Throat Swabs Routine throat swabs are collected to determine whether a patient has strep throat. If any other pathogen (e.g., Neisseria gonorrhea or Corneybacterium ditheriae ) is suspected by a physician to be causing the patient’s pharyngitis, a specific culture for that pathogen must be noted on the request slip so that appropriate culture media will be inoculated. Proper Technique for Obtaining a Throat Swab Specimen: Proper Technique for Obtaining a Throat Swab Specimen Using a tongue depressor to hold the patient’s tongue down, observe the back of the throat and tonsillar area for localized areas of inflammation (redness) and exudate. Remove a Dacron or calcium alginate swab from its packet. Under direct observation, carefully but firmly rub the swab over any areas of inflammation or exudate or over the tonsils and posterior pharynx. Do not touch the cheeks, teethe or gums with the swab as you withdraw it from the mouth. Slide 37: Insert the swab back into its packet and crush the transport medium vial in the transport container. Transport the swab to the laboratory as soon as possible. If transport will be delayed beyond 1 hour, refrigerate the swab. Wound Specimen: Wound Specimen Whenever possible, a wound specimen should be an aspirate (e.g., pus that has been collected that has been collected using a small needle and syringe assembly), rather than a swab specimen. Specimens collected by swab are frequently contaminated with indigenous microflora and often dry out before they can be processed in the CML. Slide 39: The person collecting the specimen should always indicate the type of wound infection (e.g., dog bite, post surgical or burn wound infection) on the request slip and the anatomical site from which the specimen was obtained. This provides valuable information that will enable CML personnel to inoculate appropriate types of media and be on the look out for specific organisms. GC Cultures: GC Cultures The initials GC represent an abbreviation for gonococci, a term referring to Neisseria gonorrhea. As mentioned earlier, N. gonorrhea is a fastidious bacterium that is microaerophilic and capnophilic . Only Dacron, calcium alginate or non toxic cotton swabs should be used to collect GC specimens. Ordinary cotton swabs contain fatty acids, which can be toxic to N. gonorrhea. Slide 41: When attempting to diagnose gonorrhea, swabs (vaginal, cervical, urethral, throat and rectal) should be inoculated immediately onto Thayer-Martin or Martin-Lewis medium and incubated in a carbon dioxide environment. Alternatively, they should be inoculated into a tube or bottle (e.g., Transgrow ) that contains an appropriate culture medium and an atmosphere containing 5 to 10 % co2. To prevent the loss of the co2, the bottle should be held in an upright position while inoculating. Slide 42: These cultures should be incubated at 37 degrees C overnight and then shipped to a microbiology laboratory for positive identification of N. gonorrhea. If it is necessary to transport a swab specimen, the swab should be placed into a transport medium for shipment . Never refrigerate GC swabs because the low temperature might kill the N. gonorrhea. Fecal Specimens: Fecal Specimens Ideally, fecal specimens (stool specimens) should be collected at the laboratory and processed immediately to prevent a decrease in temperature, which allows the ph to drop, causing the death of many Shigella and Salmonella species. Alternatively, the specimen may be placed in a container with a preservative that maintains a ph of 7.0 Slide 44: Because the colon is anaerobic, fecal bacteria are obligate, aerotolerant and facultative anaerobes. However, fecal specimens are cultured anaerobically only when Clostridium difficile associated disease is suspected or to diagnose clostridial food poisoning. In intestinal infections, the pathogens frequently overwhelm the normal microflora , so that they are the predominant organisms seen in smears and cultures. The Pathology Department (“The Lab”): The Pathology Department (“The Lab”) The clinical specimens just described are submitted to the CML. Within a hospital setting, the CML is an integral part of the Pathology Department . Because virtually all healthcare personnel will interact in some way(s) with the Pathology Department, they should understand how it is organized and the types of laboratory test that are performed there. The Pathology Department is under the direction of a pathologist (a physician who had an extensive, specialized training in pathology, the study of the structural and functional manifestations of disease.) Anatomical Pathology: Anatomical Pathology Most pathologist work in Anatomical Pathology, where they perform autopsies in the morgue and examine diseased organs, stained tissue sections and cytology specimens. Other healthcare professionals employed in Anatomical Pathology include cytogenetic technologists, cytotechnologists, histologic technicians , histotechnologists and pathologist’s assistants. Clinical Pathology: Clinical Pathology Clinical Pathology consists of several other laboratories: the Clinical Chemistry Laboratory or (Clinical Chemistry / Urinalysis laboratory); the hematology Laboratory or (Hematology/ Coagulation Laboratory); the Blood Bank or (Immunohematology Laboratory) In the CML of smaller hospitals, immunodiagnostic procedures are performed in the Immunology Section (sometimes called the Serology Section). Slide 48: Clinical laboratory scientist also known as medical technologists or MT’s who have 4 year baccalaureate degrees. Clinical laboratory technicians also known as medical laboratory technicians or MLT’s who have 2 year associate degrees. Organization of Typical Pathology Department: Organization of Typical Pathology Department PATHOLOGY DEPARTMENT ANATOMICAL PATHOLOGY CLINICAL PATHOLOGY MORGUE HISTOPATHOLOGY CYTOLOGY CYTOGENETICS LABORATORY ELECTRON MICROSCOPY LABORATORY CLINICAL CHEMISTRY LABORATORY HEMATOLOGY LABORATORY IMMUNOLOGY LABORATORY BLOOD BANK CLINICAL MICROBIOLOGY LABORATORY The Clinical Microbiology Laboratory: The Clinical Microbiology Laboratory CLINICAL MICROBIOLOGY LABORATORY BACTERIOLOGY SECTION MYCOLOGY SECTION MYCOBACTERIOLOGY SECTION (TB LAB) PARASITOLOGY SECTION VIROLOGY SECTION IMMUNOLOGY SECTION Responsibilities: Responsibilities The primary mission of the CML is to assist clinicians in the diagnosis and treatment of infectious diseases. To accomplish this mission, the four major responsibilities of the CML are to: Process the various clinical specimens that are submitted to the CML. Isolate pathogens from those specimens. Identify ( speciate ) the pathogens. Perform antimicrobial susceptibility testing when appropriate to do so. Slide 52: The exact steps in the processing of clinical specimens vary from one specimen type to another and also depend on the specific section of the CML to which the specimen is submitted. In general, processing includes the following steps: Examining the specimen macroscopically and recording pertinent observations (e.g., cloudiness or the presence of blood, mucus or an unusual odor). Examining the specimen microscopically and recording pertinent observations (e.g., the presence of white blood cells or microorganisms). inoculating appropriate culture media in an attempt to isolate the pathogens from the specimen and get them growing in pure culture in the laboratory. Isolation and Identification (Speciation) of Pathogens: Isolation and Identification (Speciation) of Pathogens In an effort to isolate bacteria(including mycobacteria) and fungi (yeast and molds) from clinical specimen, the specimens are inoculated into liquid culture media or onto solid culture media. The goal is to get any pathogens that are present in the specimen growing in the pure culture (by themselves) and in large number, so that there will be sufficient quantity of the organisms to inoculate appropriate identification and antimicrobial susceptibility testing systems. Bacteriology Section: Bacteriology Section In the bacteriology section, various types if clinical specimens are processed, bacterial pathogens are isolated from specimens, tests are performed to identify the bacterial pathogens and antimicrobial susceptibility testing is performed whenever it is appropriate to do so. Slide 55: Colony morphology (e.g., color, general shape, elevation, margin) Presence or absence of capsule Motility Number and location of flagella Ability to sporulate Location of spores (terminal or subterminal ) Presence or absence of various enzymes (e.g., catalase, coagulase, oxidase , urease) Slide 56: The various phenotypic characteristics (cues) useful in identifying bacteria include the following: Gram reaction (e.g., Gram-positive or Gram-negative) Cell shape (e.g., cocci , bacilli, curved, spiral-shaped, filamentous, branching) Morphologic arrangements of the cells (e.g., pairs, tetrads, chains, clusters) Growth or no growth on various types of plated media Slide 57: Ability to catabolize various carbohydrates and amino acids (miniaturized biochemical test systems” minisystems ” are often used for this purpose Ability to reduce nitrate. Ability to produce indole from tryptophan Atmospheric requirements Type of hemolysis produced Mycology Section: Mycology Section The overall responsibility of the Mycology Section of the CML is to assist clinicians in the diagnosis of fungal infections (mycoses). In the Mycology Section, various types of clinical specimens are processed, fungal pathogens and tests are performed to identify fungal pathogens. In general, the specimens processed in the Mycology Section are the types of specimens that are processed in Bacteriology Section However, three types of specimens are much more submitted to the Mycology Section than to Bacteriology Section: hair clippings, nail clippings and skin scrapings. Slide 59: A potassium hydroxide preparation (KOH prep) on hair clippings, nail clippings and skin scrapings. The KOH acts as clearing agent by dissolving keratin in the specimens. This enables the technologists to see into the specimen when they are examined microscopically and to determine whether there are any fungal elements (yeasts or molds) in the specimen. Specimens will also be inoculated onto a Sabouraud dextrose agar, aselective medium for fungi. Slide 60: Molds are identified using a combination of rate of growth and the macroscopic and microscopic observations, not by performing biochemical tests. Macroscopic observations are things that you can learn the mycelium by looking at it with naked eye-like color, texture and topography. To examine the mold microscopically, a tease mount is prepared. A drop stain is placed on a glass microscope slide. A small piece of mycelium is placed into the drop. Teasing needles (also known as dissecting needles) are used to gently pull (tease) the piece of mycelium apart. A glass cover slip is added and the tease mount preparation is examined under the microscope. Parasitology Section: Parasitology Section The overall responsibility of the Parasitology Sections is to assist clinicians in the diagnosis of parasitic diseases- specifically infections caused by endoparasites (parasites that live within the body) such as parasitic protozoa, and helminths (parasitic worms) In general parasitic infections are diagnosed by observing and recognizing various parasite cycle stages (e.g., tropho zoites and cysts of protozoa, microfilariae, eggs, and larvae of helminths )in clinical specimens. Virology Section: Virology Section The overall responsibility of the Virology Section of the CML is to assist clinicians in the diagnosis of viral diseases. Many viral diseases are diaagnosed using immunodiagnostic procedures. Other techniques used to identify viral pathogens are: Observation of intracytoplasmic or intranuclearviral inclusion bodies in specimens by cytologic or histologic examination Slide 63: Observation of viruses in specimens using electron microscopy Molecular techniques such as nucleic acid probes and polymerase chain reaction assays Virus isolation by the use of cell cultures; viruses are identified primarily by the type(s) of cell lines that they are able to infect and physical changes (called cytopathic effect or CPE) that they cause in the infected cells. Mycobacteriology Section: Mycobacteriology Section The primary responsibility of the Mycobacteriology Section or (“TB Lab”, as it is often called) of the CML is to assist clinicians in the diagnosis of tuberculosis. In the Mycobacteriology Section, various types of specimens (primarily sputum specimens) are processed, acid-fast staining is performed, mycobacteria are isolated and identified and susceptibility testing is performed. Slide 65: Mycobacterium spp. a re identified using a combination of growth characteristics (e.g., growth rate, colony pigmentation, photo reactivity and morphology) and variety of biochemical tests. Mycobaterium tuberculosis, the primary cause of human tuberculosis is a very slow-growing organism. Fortunately, the acid-fast stain enables rapid presumptive diagnosis of tuberculosis.