NUR1016 Microbiology & Pharmacology Lecture 3 (2024/2025) PDF

Tung Wah College NUR1016 AY2024/2025 Semester 2 Microbiology and Pharmacology Microbiology Lecture (L03) Principles & Methods of Diagnosis Clinical Sampling Sony SO Jan 2025  Principles & Methods of Diagnosis Clinical Sampling Cradle-to-Grave Principles Ordering...

Tung Wah College NUR1016 AY2024/2025 Semester 2 Microbiology and Pharmacology Microbiology Lecture (L03) Principles & Methods of Diagnosis Clinical Sampling Sony SO Jan 2025  Principles & Methods of Diagnosis Clinical Sampling Cradle-to-Grave Principles Ordering Collection/Sampling Transportation/Storage Specimen Processing Disposal of Diagnosis Result Reporting Treatment Interpretation Specimen Good Quality Clinical Specimen Collection/Sampling Principles (1) Compliance with Statutory Requirements, Standard Operating Procedures (SOPs), & Professional Standards (CLSI, 2017; PHLC, 2025; WHO, 2020)  GOOD QUALITY Clinical Specimen Collection/Sampling Principles Collection/Sampling (PHLC, 2025)  Good Quality Clinical Specimen Collection/Sampling Principles – Adherence to Aseptic Non Touch Technique (ANTT) Requirements 無菌不接觸技術 o Aseptic technique: the method by which precautions are taken during invasive clinical procedures to prevent the transfer of microorganisms from the health care practitioner, procedure equipment or the immediate environment to the patient. Regardless of the setting, the aim is always to prevent the transfer of pathogenic microorganisms into or onto the patient. Key-Part o Non-touch technique (NTT): An integral component in achieving asepsis whereby Key-Parts and Key-Sites avoid contamination; if Key-Parts and Key- sites are required to be touched, sterile gloves should be used to carry out the procedure. Key-site o Aseptic non-touch technique (ANTT®): a specific type of aseptic technique with a unique theoretical and practice framework that combines the aseptic technique, and non-touch techniques. Aseptic / asepsis: free from pathogenic organisms. (The Association for Safe Aseptic Practice, n.d.) Clinical Specimen Collection/Sampling Procedures (1 of 3) Use Appropriate Specimen Containers/Sampling Devices Collection/Sampling Types of Clinical Specimen Containers (PHLC, 2025) 【瑪麗醫院肝移植病人疑抽血時染丙型肝炎 器官衰竭亡】 【袁國勇料輔助抽血器受污染 醫管局將改用一次性輔助抽血器】 https://www.youtube.com/watch?v=egWg9WsLr0k (Newsline Express, 2019)  Good Quality Clinical Specimen Collection/Sampling Principles (5) – Aware of The Usage of RIGHT Clinical Specimen Containers Midstream Urine (MS) container Blood Sampling Holder + with boric acid Winged Needle Stool Collection Cup with Small Spoon clean catch urine sputum container container Vacuum Blood Collection Tubes (PHLC, 2025) Good Quality Clinical Specimen Collection/Sampling Principles (5) – Aware of The Usage of Clinical Specimen Containers Invented by COPAN, flocked swabs are a special type of swab produced with an applicator tip that is spray-coated with short Nylon® fibers arranged perpendicularly (COPAN, 2024) Clinical Specimen Collection/Sampling Procedures (2 of 3) Sampling Procedures Collection/Sampling Microbiological Tests Used In The Diagnosis Of Tuberculosis (TB) o Sputum for acid fast bacilli (AFB) direct smear and culture examination o Mycobacterial culture -6-8 weeks o Other clinical samples for microbiological diagnosis include gastric washing, bronchial aspirate, pleural fluid, biopsy specimens of the lung o or non-respiratory specimens like early morning urine (EMU), lymph node aspirate, pericardial fluid, cerebrospinal fluid, joint fluid, etc. o EMU for 3 consecutive mornings - 1 litre bottle EMU - The first urine after a night‘s rest (Au et al., 2006) Specimen Collection – collect Urine from Urinary Catheter Use alcohol swabs to disinfect sampling port or the urine bag outlet Catheter Specimen Urine (CSU) - Catheter Specimen Urine (CSU) - Collect Urine After Catheter Insertion from Catheter Sampling Port From Urine bag Specimen Collection – Collect Urine by Midstream Urine Midstream Urine (MSU) - Urine For Culture & Sensitivity Tests (C&ST) – A urine culture is a test that checks your urine for bacterial or fungal (yeast) infections ie diagnosing a urinary-tract infection (UTI) in adults General Instructions o Before procedure – perform hand hygiene. o Clean urethral opening and genital area with a wet tissue. Keep foreskin retracted (Male) or labia apart (Female), perform hand hygiene again. o Do not tough the inside of the container or container cap. o Pass the first portion voided urine into toilet and collect midstream urine into MSU specimen container to 1/2 full. Midstream Urine (MS) container o Seal the container and label appropriately. with boric acid (preservatives) o Deliver specimen to Clinical Laboratory immediately. Store specimen at 4 - 8oC if not deliver immediately. (Chan & Hou Medical Laboratories Ltd, n.d.; Pathology Department St. Paul's Hospital, 2023) First Catch/ Void Urine / Clean Catch Urine General Instructions Clean Catch Urine o Clean urethral opening and genital area with a wet Urinary Antigen Tests (UAT) tissue. Keep foreskin retracted (Male) or labia apart (Female), perform hand hygiene again. Urinary Legionella Antigen Test Legionella pneumophila o Do not tough the inside of the container or container cap. Pneumococcal Urinary Antigen Test o Collect first catch urine into specimen plain container Streptococcus pneumoniae ie without preservatives to 1/3 plain specimen o Seal the container and label appropriately. Test for Chlamydia & Neisseria by PCR container o Please send specimen together with request form to the Clinical Laboratory. If specimen cannot be delivered immediately, please keep in a cool place. (Lo, n.d.; St. Paul's Hospital Pathology Department, 2023) Clinical Specimen Collection/Sampling Procedures (3 of 3) Skin Disinfection For Sterile Sites Collection/Sampling Specimen Collection – Skin Disinfection For Sterile Sites https://www.youtube.com/watch?v=LZqiu6PKrZU https://www.youtube.com/watch?v=uNiYPBMBdtQ&t=315s (BioMérieux University, 2025; Design Science, 2018) Skin Disinfectants & Time Required 聚维酮碘 (Povidone-iodine PVP-I) + 70% Alcohol Solution = Skin Disinfection Time 1.5 to 2 minutes + 30 seconds = 2.5 minutes 碘酊(tincture iodine) + 70% Alcohol Solution = Skin Disinfection Time 30 seconds + 30 seconds = 1 minute Iodine-containing preparations 70% Alcohol Solution 30 seconds for tincture of iodine 30 seconds 1.5 to 2 minutes for iodophors - (Povidone-iodine) 酒精紙 Alcohol pad (CLSI, 2017; WHO, 2010;中华人民共和国国家卫生健康委员会, 2020)  Skin Disinfectants & Time Required – Alcoholic CHG 2% w/v chlorhexidine gluconate (CHG) and 70% v/v isopropyl alcohol (IPA) Chlorhexidine Gluconate (CHG) 葡萄糖酸氯己定 3M SoluPrep Swab BD ChloraPrep SEPP BD ChloraPrep BD ChloraPrep Frepp Disinfection time - Dry Site = 30s (3M, n.d.) Skin Disinfection Time 衛生行業標準 《WS/T 661—2020 靜脈血液標本採集指南》 以穿刺點為圓心，以圓形方式自內向外進 行消毒，消毒範圍直徑5cm，消毒2次。消 毒劑發揮作用需與皮膚保持接觸至少30s， 待自然乾燥後穿刺，可防止標本溶血及灼 燒感。如靜脈穿刺比較困難，在消毒後需 要重新觸摸血管位置，宜在採血部位再次 消毒後穿刺。 30 (中华人民共和国国家卫生健康委员会, 2020) Safe Transportation of Clinical Specimen Clinical Specimen Collection/Sampling 樣本收集箱一度遺失 醫管局致歉 Transporation Accidents (4.9.2020) ISD, Hong Kong SAR Government 香港特區政府新聞處 東華東院遺六病人血液、尿液樣本於收集箱 未依時化驗 – 20201120 – 港聞 - 有線新聞 CABLE News (On.cc, n.d.) Good Quality Clinical Specimen Collection/Sampling Principles (7) –- Safe Transportation of Collected Specimen Principles Compliance to Requirements – Local Laboratory, international standards Required Temp. Cooler / Room Temp 溫度限規保冷/室溫 Vertical Required Time 垂直放置 時間規限 Protect Transport Container Affixed from light With Biohazard Label 遮光需要 運送容器貼有生物危險告示 (PHLC, 2025; WHO, 2021)  Specimen Transportation – Temperature Requirement Specimens should be transported to the laboratory as soon as possible, preferably within the same day after collection. In general, if delay in despatch is unavoidable, keep specimens refrigerated at 40C Except the following Guide to Requests for Specimens for bacterial / fungal culture and parasitology examination should be kept at room temperature: Laboratory Testing o Blood o Cerebrospinal fluid o Dermatological specimens o Genital specimens o Spore strips o Enteric specimens EDTA blood for hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA and human immunodeficiency virus (HIV-1) RNA quantitation o must be kept at 2-250C and arrive at PHLC within 24 hours of collection. Public Health Laboratory Services Branch o Otherwise, plasma/serum should be separated within Centre for Health Protection 24 hours of collection, stored at 2-80C and sent to Department of Health The Government of the Hong Kong Special Administrative Region PHLC within 6 days. (CHP, n.d.) Good Quality Clinical Specimen Collection/Sampling Principles (7) – Adherence to Safety Transportation - Local Requirements (HA Central Committee on Infectious Diseases, 2006;衞生署衞生防護中心公共衞生化驗服務處, 2009) Good Quality Clinical Specimen Collection/Sampling Principles (7) – Adherence to Safety Transportation - International Requirements- Air Transportation The International Air Transport Association (IATA) (Gruber, 2010) Good Quality Clinical Specimen Collection/Sampling Principles (3) – Adherence to Aseptic Non Touch Technique (ANTT) Requirements Keep lid of culture Heat Sterile Reusable Proper Pipetting Proper Laboratory Attire plate closed Inoculating Loop (PHLC, 2008;WHO, 2020)  Biosafety levels (BSL) o are used to identify the protective measures needed in a laboratory setting to protect workers, the environment, and the public. o The four biosafety levels are BSL-1, BSL-2, BSL-3, and BSL-4 (maximum level of containment Ebola virus, Variola virus (smallpox) Mycobacterium tuberculosis Influenza, Salmonella infection Escherichia coli (IBC, n.d.) Biological Safety Cabinet (BSC) All Class II biological safety cabinets use filters and controlled airflows to capture contamination, designed to protects the operator, product, and environment. https://www.youtube.com/watch?v=zJ7PCCLPpfk https://www.youtube.com/watch?v=S6Zs483R5SM Fundamentals of Working Safely in a Biological Safety Thermo Scientific class II biological safety cabinet Cabinet (BSC): Factors Affecting BSC Airflow animation How To Identify Unknown Microbes? Specimen Processing  How To Identify Unknown Microbes? Methods microbiologists use to identify microbes (mostly bacteria) to the level of genus and species fall into three categories: Phenotypic o Consideration of morphology (microscopic and macroscopic) as well as microbial physiology or biochemistry Biochemistry Features antibody Immunologic Genotypic o Involve serological analysis o Analysis of microbe’s DNA or RNA antigen o Analysis of microbe using antibodies o Increasingly being used as a sole o Analysis of patient’s antibodies using resource for identifying microbes prepackaged antigens Phenotypic methods Phenotype refers to the traits (特質) that an organisms is expressing in the present Involve examining microbe appearance (外觀) and behavior (行為) - Appearance: Microscopic appearance (shape and arrangement) - Behavior: Fundamental chemical characteristics such as (1) nutrient requirements, (2) products given off during growth, (3) presence of enzymes, (4) mechanisms for deriving energy, and (5) chemical composition of its walls and/or membranes - What types of enzymatic activities it can carry out? - What kind of physical conditions it thrives in? - What antibiotics it is susceptible to? Identify Bacteria By Staining o Gram Stain o Ziehl-Neelsen Stain Identify Microbes By Microscopy Completed in last week Identify Microbes By Microbiological Culture Specimen Processing  Blood Culture Medium Blood culture: blood specimen submitted for culture of microorganisms. It enables the recovery of potential pathogens from patients suspected of having bacteremia or fungemia. BD BACTEC Plus BD BACTEC Plus BD BACTEC BD BACTEC Standard BACTEC Myco/F Lytic Aerobic medium Anaerobic medium Standard Aerobic medium Anaerobic medium culture bottle contains resins for antibiotic neutralization (BD Becton, Dickinson and Company, n.d.) Blood Culture Videos https://www.youtube.com/watch?v=GDji2lQGiJI Genotypic Methods Specimen Processing Genotypic Methods o Examining the genetic material itself (DNA and/or RNA) o Many advantages over phenotypic methods Culturing of the microorganisms is not always necessary – many microorganisms we cannot grow in lab Genotypic methods are increasingly automated, producing rapid results that are often more precise than phenotypic methods Numerous viable nonculturable (VNC) microbes are currently being identified by genotypic methods What are DNA & RNA? Deoxy-ribonucleic Acid (DNA) Ribonucleic acid (RNA) Nucleic acids 核酸 -two main types of nucleic acids are Ribonucleic acid (RNA) & Deoxyribonucleic Acid (DNA) DNA/RNA Components Nucleotides 核苷酸 - consists of a nitrogenous base, a five-carbon Nucleosides 核苷 sugar, and one or more phosphate groups. - consists of a nitrogenous base and a five-carbon sugar (ribose or deoxyribose) - Basic Elements – Carbon, Nitrogen, Oxygen, and Phosphorous nitrogenous base AUCG o Ribose has one more OH group than a deoxyribose molecule. o The word de-oxy infers that ribose has lost an oxygen atom. o The missing OH group makes this five carbon sugar more stable than an RNA molecule. DDS nitrogenous base ATCG What is Deoxyribonucleic Acid (DNA)? What is Deoxyribonucleic Acid (DNA)? o Deoxyribonucleic Acid (abbreviated DNA) is the molecule that carries genetic information for the development and functioning of an organism. o DNA is made of two linked strands (雙鏈)that wind around each other to resemble a twisted ladder — a shape known as a double helix. o Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups o Attached to each sugar is one of four Nitrogenous Bases: Adenine (A) Phosphate Group Thymine (T) Nitrogenous Base Cytosine (C) DNA Guanine (G) Nucleotide sugar molecule (deoxyribose) (National Human Genome Research Institute, 2023)  What is Ribonucleic acid (RNA)? o Ribonucleic acid (abbreviated RNA) is a nucleic acid present in all living cells that has structural similarities to DNA. o Unlike DNA, RNA is most often single-stranded (單鏈). o An RNA molecule has a backbone made of alternating phosphate groups and the sugar ribose, rather than the deoxyribose found in DNA. Attached to each sugar is one of four bases: Adenine (A) Phosphate Group Uracil (U) Nitrogenous Base Cytosine (C) RNA (A, U, C, or G) Guanine (G) Nucleotide sugar molecule (ribose) (National Human Genome Research Institute, 2024) Examples of DNA Examples of RNA viruses are viruses are herpes, influenza, SARS, MERS, COVID-19, smallpox, hepatitis B, Dengue virus, hepatitis C, adenoviruses, and hepatitis E, West Nile fever, Ebola warts virus disease, rabies, polio, mumps, and measles. RNA viruses have a higher mutation rate when compared to DNA viruses. Also, their genetic diversity makes it difficult to produce effective vaccines against them. RNA differs from DNA chemically in two respects: 1. the nucleotides in RNA are ribonucleotides—that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose; 2. RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) (ThoughtCo, 2013) Commonly Used Genotypic 基因型 Tests o Polymerase Chain Reaction (PCR) o Real-Time PCR Specimen Processing  Polymerase Chain Reaction (PCR) PCR ( 聚 合 酶 連 鎖 反 應 ) results in the production of numerous identical copies of DNA or RNA molecules within hours - Can amplify even minute quantities of nucleic acids present in a sample - Using primers to amplify specific DNA sequences https://www.youtube.com/watch?v=5s50wWkq4Hk What Real-Time PCR? https://www.youtube.com/watch?v=iu4s3Hbc_bw Real-Time PCR o (also known as qPCR; q = quantitative) o Use fluorescent labeling during the amplification procedure and the level of fluorescent is measured in real time as the reaction is running o Fully automated and faster than traditional PCR o Measurement of the Ct value – Relative measure of the concentration of target in the PCR reaction o The PCR Ct (cycle threshold) value refers to the number of cycles needed to replicate enough DNA/RNA to be detected (crosses a threshold line). o A Ct value of 20 means it took fewer cycles to produce enough DNA/RNA than a Ct of 30. o The lower Ct value means there was more DNA/RNA in the sample to begin with. Real-Time PCR and Ct value (cycle threshold) Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold The smaller the Ct value, the larger the quantity of the sample Significance of Ct value Ct < 35: Confident positive results Ct > 35: Can be false positive results COVID-19:  Immunological Test Antigen – Antibody Reaction Antibody Antigen Antibody  Immunological Tests o Characteristics of antibodies (such as their quantity or specificity) can reveal the history of a patient’s contact with microorganisms or other antigens ie serological testing o Serological testing is based on the principle that antibodies have extreme specificity for antigens When a particular antigen is exposed to its specific antibody, it will fit like a hand in a glove This interaction can be visualized macroscopically or microscopically Used to determine the immunologic status of patients, confirm a suspected diagnosis, or screen individuals for disease  Antigen & Antibiotic Tests Serologic Test - test to detect the presence of antibodies against a microbes. A serologic test can determine whether a person has been exposed to a particular microbes The molecular basis of immunological testing is the binding of an antibody (Ab) to a specific site (epitope) on an antigen (Ag) https://www.youtube.com/watch?v=PkczTPfIE20  Antigen-Antibody Reaction Antigen (抗原) is anything that is foreign to the human body. It can be a virus, it can be a bacteria, and in some cases your own body will appear as foreign. And so you can have in certain instances where your own body will make antibodies against parts that are part of you. Antibody (抗體) is a part of the host cell's defense. It's made by a certain type of white blood cell that's called a B cell. Antigen An antibody is a protein component of the immune system that circulates in the blood, recognizes foreign substances like bacteria and viruses, and neutralizes them. After exposure to a foreign substance, called an antigen, Light light antibodies continue to circulate in the blood, providing protection against chain chain future exposures to that antigen. The structure of the antibody consists of two light chains and two heavy chains, and at the very tip of the antibody is a hypervariable region, and this hypervariable region allows the antibody to make different types of antibodies that will respond to all of the antigens that will assault the body. heavy chains (National Human Genome Research Institute, 2024) Got Vaccinated = Got Antibody = Got Protection? https://www.youtube.com/watch?v=01c8ui2hpNg&t=71s Neutralizing Antibodies, Antibody Titer o Neutralizing Antibodies refer to those antibodies that have the ability to dissolve and reduce the infectivity of viruses. These antibodies can block the virus from entering human cells to prevent infection. Neutralizing antibodies are mainly consisted of IgA, IgG, and IgM, among which IgG is the most abundant and multifunctional component of neutralizing antibodies o Antibody Titer - blood test is done to determine the presence (qualitative) and amount (quantitative) of antibodies in the blood. ▪ to determine if a person was infected by any pathogen in the past. ▪ requirement of a booster dose.  What Do IgG, IgM? o Antibodies, also called immunoglobulins (Ig), are important elements of the human immune system. Common antibodies include immunoglobulin M and immunoglobulin G, namely IgM and IgG. o When the human body is invaded by bacteria or viruses, the body will first produce IgM antibodies as the first line of defense, but they are usually short- lasting; then the body will produce IgG as a long- lasting protection. Therefore, IgM positivity usually means "currently infected" or Antibody detection: "recently infected"; - IgM: Usually 5-7 days after onset of illness IgG positivity usually means "previously infected" - Antibody titre: Paired acute and convalescent or "prevailing immunity". sera preferably 10-14 days apart - IgG: Any time after window period for persistent infection and immunity testing Understanding Hepatitis B Serological Tests Results o HBsAg (Hepatitis B surface antigen) - positive result indicates that a person is infected with hepatitis B. This test can detect the presence of the hepatitis B virus (called the “surface antigen”) in the blood. If a person tests “positive,” then further testing is needed to determine if this is a new “acute” infection or a “chronic” hepatitis B infection. o anti-HBs or HBsAb (Hepatitis B surface antibody) – positive result indicates that a person is protected against the hepatitis B virus. This protection can be the result of receiving the hepatitis B vaccine or successfully recovering from a past hepatitis B infection. A positive anti-HBs (or HBsAb) test result indicates that a person is “immune” and protected against the hepatitis B virus and cannot be infected. o anti-HBc or HBcAb total (Hepatitis B core antibody) – positive result indicates that a person has been exposed to the hepatitis B virus at some point during their life. A positive HBcAb test alone cannot tell if a person has a past or current hepatitis B infection. The core antibody does not provide any protection against the hepatitis B virus (unlike the surface antibody described above). This test can only be fully understood by knowing the results of the first two tests (HBsAg and anti-HBs). (Hepatitis B Foundation, n.d.) Understanding Hepatitis B Serological Tests Results (Hepatitis B Foundation, n.d.)  Enzyme-linked Immunosorbent Assay (ELISA) What is Substrate? A substance to which another substance is applied we call it as a substrate. The enzyme-linked immunosorbent assay Step 4 - Finally, a substrate is added to the (ELISA) is an immunological assay commonly plate. ELISA assays are usually chromogenic (顯 used to measure antibodies, antigens, proteins and glycoproteins in biological 色的) using a reaction that converts the substrate samples. Some examples include: diagnosis of into a coloured product which can be HIV infection measured using a plate reader. Step 3 - detection antibody is added. This antibody is labelled with an Step 2 - sample (e.g. urine, serum, or cell enzyme, usually horse radish supernatant) with any antigen is added. peroxidase or alkaline phosphatase. Detection antibody binds to any target antigen will bind to the capture antibody antigen already bound to the plate. already coating the plate. Samples are usually added in duplicate or triplicate (to allow for statistical analysis), and in varying concentrations to guarantee it falls within the levels of detection of the assay. Step 1 - to coat the ELISA plate with capture antibody, any excess, unbound Again any excess sample is washed from the antibody is then washed from the plate. The capture antibody is an antibody raised plate against the antigen of interest. (Horlock, n.d.) Sandwich ELISA Protocol https://www.youtube.com/watch?v=ojYHnZZbwz8  False Positive Result 驗錯孕婦感染愛滋病 仁安醫院致歉 發佈日期: 2016-05-10 21:00 港澳 仁安醫院發言人就事件對當事人帶來困擾及不便致歉，指醫護 人員安排轉介事主到威院跟進，以及該測試或會出現0.1%假 陽性結果，即未有感染病毒但結果顯示呈陽性反應。 醫生︰或未有覆檢結果所致 婦產科專科醫生靳嘉仁表示， 20年前他在公立醫院任職期間， 曾遇上類似案例，孕婦跟丈夫互相質疑對方先感染愛滋病毒。 仁安醫院為驗錯一名孕婦感染愛滋病 兩星期後再一次測試，才確認兩人並無染病，成為唯一一次產 毒致歉。 懷孕四個多月的34歲女事主 前抽血報告結果呈假陽性的個案。 上月到仁安醫院產前檢查，其後驗血 報告顯示她對愛滋病毒呈陽性反應。 血液科專科醫生馬紹鈞指部分醫院驗出呈陽性結果後，會將樣 院方拒絕她的分娩預約，只轉介她到 本送往衛生署覆檢，但並非每間醫院都有類似做法。他相信今 威爾斯醫院覆驗，證實對愛滋病毒呈 次事件，乃化驗員未有再確認假陽性結果所致。 陰性，仁安醫院早上向事主道歉。 https://www.thenewslens.com/article/29156 Serological Tests – Specificity 特異度Vs Sensitivity 靈敏度 1. Sensitivity: The detection of even minute quantities of antibodies or antigens in a specimen - A test with high sensitivity will have a low false-negative rate 2. Specificity: The property of a test to focus on only a certain antibody or antigen and not to react with unrelated or distantly related ones - A test with high specificity will have a low false-positive rate (Virtual AIDS Office, n.d.) HIV Laboratory Diagnostic Algorithm o Enzyme-linked Immunosorbent Assays (ELISAs) for HIV screening. ELISA is a highly sensitive test, and can sometimes generate false positives by mistaking other antibodies for those of HIV. Positive or o At present, the most common strategy for HIV antibody equivocal testing uses a highly sensitive ELISA followed by a confirmatory test with Western Blot, HIV-1/HIV-2 antibody differentiation immunoassay, molecular test o Using this strategy, the achievable false positive rate can be as low as 38.0°C or > 100.4°F) Organism identified from blood following : Leukopenia (≤ 4000 WBC/mm3 ) or leukocytosis Organism identified from pleural fluid New and persistent or Progressive (≥ 12,000 WBC/mm3) and persistent Positive quantitative culture or corresponding semi- For adults ≥ 70 years old, altered mental status quantitative culture result from minimally- Infiltrate with no other recognized cause contaminated LRT specimen (specifically, BAL, Consolidation protected specimen brushing, or endotracheal Cavitation aspirate) And at least one of the following: Pneumatoceles, in infants ≤1 ≥ 5% BAL-obtained cells contain intracellular bacteria New onset of purulent sputum or change in on direct microscopic exam (for example, Gram’s stain) year old character of sputum, or increased respiratory Note: In patients without secretions, or increased suctioning requirements Positive quantitative culture or corresponding semi- underlying pulmonary or cardiac quantitative culture result of lung tissue Dyspnea, or tachypnea , or new onset or disease (such as respiratory worsening cough Histopathologic exam shows at least one of the distress syndrome, following evidences of pneumonia bronchopulmonary dysplasia, Rales or bronchial breath sounds Abscess formation or foci of consolidation with pulmonary edema, or chronic Worsening gas exchange (for example, O2 intense PMN accumulation in bronchioles and obstructive pulmonary disease), desaturations [for example, PaO2/FiO2 ≤ 240] alveoli one definitive chest imaging test increased oxygen requirements, or increased result is acceptable. ventilator demand Evidence of lung parenchyma invasion by fungal hyphae or pseudohyphae (NHSN, 2024) Catheter associated Urinary Tract Infection (CAUTI) in any age patient Patient must meet 1, 2, and 3 below: 1. Patient had an indwelling urinary catheter that had been in place for more than 2 consecutive days in an inpatient location on the date of event AND was either: Present for any portion of the calendar day on the date of event† , OR Removed the day before the date of event‡ 2. Patient has at least one of the following signs or symptoms: fever (>38.0°C) suprapubic tenderness* costovertebral angle pain or tenderness* urinary urgency ^ urinary frequency ^ dysuria ^ 3. Patient has a urine culture with no more than two species of organisms identified, at least one of which is a bacterium of ≥105 CFU/ml (See Comments). All elements of the Symptomatic Urinary Tract Infection (SUTI) criterion must occur during the Infection window period (IWP) ). *With no other recognized cause (see Comments) ^ These symptoms cannot be used when catheter is in place. An IUC in place could cause patient complaints of “frequency” “urgency” or “dysuria”. (NHSN, 2024)  Determining Clinical Significance Of Cultures o Cultures may be obtained from sites that are either colonized with bacteria, and those colonized bacteria will increase the risk of contamination and may lead to false positive results Sites that are well known for contamination include sputum and nasal passages. Poor culture-collection technique may also increase the risk of contamination. o Sites that are typically thought of as sterile include cerebral spinal fluid, blood, and pericardial fluid. Even implementing stringent infection prevention and control requirements, the overall blood culture contamination rate is still maintaining around 1% to 3% ie false positive; If cultures are drawn post-antibiotic administration, there may be a decrease in the blood-culture yield ie may cause false negative. o Therefore, in general, diagnosis of clinical infection should not only refer only to positive culture results, but also refer to Imaging Test Evidence, clinical signs & symptoms, and other laboratory test results e.g. WBC count in urinalysis, eg- Asymptomatic bacteriuria refers to the isolation of bacteria in urine culture from a patient without signs or symptoms of urinary tract infection (UTI). A positive urine culture result (with or without pyuria  WBC in urine) alone does not meet criteria for initiation of antibiotics according to infectious diseases guidelines (CDC, SIDP & ASHP, n.d.; CDC, n.d.; Giuliano, Patel & Kale-Pradhan, 2019) Take Home Message Principles & Methods of Diagnosis Clinical Sampling Cradle-to-Grave Principles So, How? o Take GOOD QUALITY specimen Use right sampling methods Collect from right patient at the right time Transport specimen in the right parameters setting o Process specimen by right microbiological testing methods o Prompt right reporting o Right interpretation o Right treatment o Optimizing patient care The End Click Here for reference list