Learn: MMSC 438 Exam 3

STI epidemiology

-increased cases of Chlamydia, Gonorrhea and Syphillis in the US in recent years

-coinfection with Chlamydia and Gonorrhea is common

male genital tract normal biota

-CoNS

-corynebacterium spp.

-mycobacterium smegmatis

female genital tract normal biota (pre-puberty and post-menopausal)

-staphylococci

-corynebacterium spp.

female genital tract normal biota (reproductive age)

-lactobacilli are predominant

predisposed to STI

-sexually active

-multiple sexual partners

-unsafe sexual practices (no contraceptives, etc.)

-medical devices (IUD, etc.)

STI transmission

organisms are passed from an infected individual or asymptomatic carrier to an uninfected individual

-sexual contact: organisms enter mucosal or squamous epithelium of the vagina, cervix, rectum or oropharynx

-vertical transmission: organisms spread from mother to fetus during pregnancy or delivery

-blood: pathogens in genital tract get into circulation

-genital secretions: contain pathogens

urethritis and cervicitis (causative agents)

-chlamydia trachomatis (serovars D to K)

-neisseria gonorrhea

-mycoplasma hominis

-mycoplasma genitalium

-ureaplasma urealyticum

-streptococcus agalactiae

genital discharge (causative agents)

-candida albicans

-trichomonas vaginalis

-gardnerella vaginalis and mobiluncus

genital ulcers (causative agents)

-herpes simplex virus

-treponema pallidum

-klebsiella granulomatis

-haemophilus ducreyi

-chlamydia trachomatis (serovars L1, L2 and L2)

genital warts and cancer (causative agents)

-human papillomavirus (HPV)

immunodeficiency disease (causative agents)

-human immunodeficiency virus (HIV) 1 and 2

chlamydia trachomatis (serovars D to K)

-mucopurulent vaginitis, urethritis and cervicitis

-nongonococcal urethritis (NGU)

-pelvic inflammatory disease (PID) if left untreated

-asymptomatic infections are common, more common in women

-most commonly transmitted STI in the US

-OIP that cannot be seen on gram stain because it has to be inside of human cells

mycoplasma hominis, mycoplasma genitalium, ureaplasma urealyticum

-mucopurulent vaginitis, cervicitis and urethritis

-nongonococcal urethritis (NGU)

-bacteria have no cell wall, so ß-lactam drugs are not an effective treatment

neisseria gonorrhoeae (diseases)

-mucopurulent vaginitis, urethritis, and cervicitis

-*gonococcal urethritis* (gonorrhea)

-PID: if untreated

-disseminated gonococcal infection (DGI): untreated infection gets into circulation leading to rash and septic arthritis

-asymptomatic infections common in women

-FIP, may be inside or outside of human cells

neisseria gonorrhoeae (identification)

-gram negative diplococci (tends to be intracellular, FIP)

-oxidase +

-superoxol +

-glucose + (only uses 1 carbohydrate)

-NAAT is best diagnosis

pelvic inflammatory disease (PID)

sequelae to untreated genital infections in females

-untreated infection with Neisseria gonorrhea or Chlamydia trachomatis

-bacteria ascends into the fallopian tubes, uterus and ovaries leading to scarring and inflammation

-may lead to infertility

bacterial vaginosis (BV)

-a decrease in the normal biota of the vagina (lactobacilli) causes the pH of the vagina to increase

-abnormal vaginal flora (Gardnerella vaginalis and mobiluncus) overpopulate

-leads to foul "fishy" smelling gray vaginal discharge

-*NOT sexually transmitted*

nugent grading system

a system to diagnose bacterial vaginosis in which bacterial smear is viewed under oil immersion to count organisms and grade

-normal conditions (low/ 0 score): > 30 lactobacilli, 0 mobiluncus, 0 gardnerella

-score of 0-3= normal biota

-score of 4-6= altered vaginal flora, repeat test

-score of 7-10= bacterial vaginosis

amsel scoring

a system to diagnose bacteiral vaginosis based on presence of symptoms

-clue cells (epithelial cells covered in gardnerella vaginalis)

-vaginal pH > 4.5

-discharge

-odor in Whiff Test

herpes simplex virus (type 1 and 2)

-genital herpes: ulceration in the genitals, buboes found in the inguinal region

-ulcers go away after 7-10 days but virus remains latent in the nerves until reactivated

-reactivation causes a flare up that leads to formation of more ulcers

treponema pallidum (subsp. pallidum)

-primary stage: formation of painless chancre ulcer that goes away after 1-2 weeks

-secondary stage: rash forms on the palms and feet, then goes away on its own

-latent stage: no symptoms

-tertiary stage: in ~30% of people an untreated infection will disseminate and infect secondary organs, e.g. heart, CNS

Klebsiella granulomatis

-granuloma inguinale (donovanosis): formation of painless ulcers in the genitals

-wright or giemsa stain shows formation of donovan bodies (macrophages containing blue rods with polar granules)

-rare infection in the US

Haemophilus ducreyi

-chancroid: formation of painful ulcers on the genitals, with buboes in the groin area

-rare in the US

chlamydia trachomatis (serovars L1, L2 and L3)

-lymphogranuloma venereum (LGV): painless ulcers form on the genitals with buboes in the groin and genital elephantiasis

-VERY rare infection in the US

human papillomavirus (HPV)

-genital warts: formation of warts caused by E6 and E7 viral proteins causing uncontrolled abnormal cell growth

-cervical cancer, anogenital cancer: cancer cells form due to E6 and E7 viral proteins that cause uncontrolled cell proliferation

-Cevarix or Gardasil vaccines available for prevention

human immunodeficiency virus (HIV type 1 or 2)

-sexually transmitted virus that penetrates the mucosal epithelium, attaches to CD4+ T helper cells and destroys the immune response

-lack of T helper cells leads to lack of B cell activation so there is no humoral (antibody) or cellular immune response

-over time the immune system becomes completely depressed and leads to strange/ opportunistic infections not seen in immunocompetent individuals

-acute HIV infection: right after infection occurs the virus is present and killing T helper cells

-clinical latency: virus incorporates itself into genome of host cells (via reverse transcriptase)

-symptomatic AIDS related complex (ARC)

-AIDS: full immunosuppression, lots of opportunistic infections

AIDS related infections

-mycobacterium avium complex infections

-candidiasis (esophageal and vulvovaginal)

-pneumocystis pneumonia

-fungal and parasitic infections: aspergillosis, histoplasmosis, cryptococcsis, cryptosporidosis, toxoplasmosis

-viral infections: CMV, HSV, HPV, varicella zoster virus

-condolymata acuminata: anogenital warts caused by HPV

-mollusum contagiosum: benign wart-like tumors on the skin

-proctitis: inflammation of the prostate (STI)

neonatal STI transmission

-vertical transmission: transmission from mother to fetus/ newborn

-congenital transmission: maternal infection crosses the placenta and infects the fetus

-perinatal infection: infant passing through the birth canal becomes infected, or infection occurs immediately postpartum

neonatal STI

-symptoms: low birth weight, meningitis, sepsis

-streptococcus agalactiae (GBS): normal biota in the mother's vagina inocculates infant during birth and leads to meningitis or sepsis

-STI pathogens: Treponema pallidum, Chlamydia trachomatis, Neisseria gonorrhea, Mycoplasma and Ureaplasma spp.

-viruses and parasites: herpes simplex virus, CMV, HPV, toxoplasma gondii

TORCH serology panel

serologic test that is used on newborns to screen for infection with

-Toxoplasma gondii

-Rubella

-CMV

-HSV

group B strep screening

between 35-37 weeks gestation a woman should be tested for Streptococcus agalactiae in her normal biota, and treat her if she is

-swab vaginal and rectal area

-inoculate swab into lim broth

bacterial STI diagnosis

-culture

-gram stain: morphology is highly indicative of what pathogen you have

-nugent grading/ amsel scoring

-fluorescent antibody test: Chlamydia and Treponema pallidum detection in patient sample

-darkfield microscopy

-antigen detection: immunoassays, ELISA, CT antigen pgp3

-molecular tehcniques: DNA probes, Affirm VP III, NAAT

-syphilis serology

fungal STI diagnosis

-gram stain

-10% KOH wet prep: yeasts

-culture

-DNA hybridization

viral STI diagnosis

-viral detection: gene probes, PCR

-serology

parasitic STI diagnosis

-saline wet mount: trichomonas vaginalis motility

-DNA hybridization techniques

child abuse lab implications

-any STD diagnosed in a child is implicated to sexual abuse

-all isolates that are identified need to be documented for legal purposes

-chain of custody of child's sample needs to be proven

genital specimen collection, transport and storage

-avoid contamination: lots of normal biota in the area can make it difficult to recover pathogen

-rayon tipped swabs: used for mycoplasma, chlamydia and neisseria gonorrhoeae

-obtain human cells for chlamydia (OIP)

-dracon swabs: used for viral specimens

-avoid swabs with calcium alginate or cotton (fatty acids) that damage specimen

-transport swab in media with charcoal or albumin to neutralize toxic substances that may damage pathogen

-transport ASAP

-do not refrigerate (cold sensitive)

-bedside plating is best if possible

chlamydia and gonorrhea

common coinfection

-when screening for STIs always screen for both because they are common and often found together

herpes simplex virus screening

-obtain vesicle fluid from ulcer with dracon swab

-perform Tzanc smear to look for giant multi-nucleated cells

-perform NAAT to confirm diagnosis

genital specimen types

-urethral, vaginal discharge or endoverical swabs

-suction curette of endometrium

-PID: peritoneal fluid, cul de sac material, of fallopian tube/ ovarian sample

-anal culture (N. gonorrhoeae)

-aspirates: good for anaerobic culture

-scrapings/ biopsies

-lesion vesicle fluid (syphillis, herpes)

-IUD: submit entire device in sterile container

-urine or ejaculate for prostatic secretions

genital specimens for anaerobic culture

-cervical aspirate

-uterus/ endometrium sample

-fallopian tube sample

-ovary sample

-placenta from C-section

neisseria gonorrhoeae culture media

-JEMBEC plate: bedside plating

-nonselective media: chocolate agar

-modified thayer martin (MTM)

-martin lewis

-NYC agar

-GC lect

chlamydia and mycoplasma transport media

-transport: 2SP media

-refrigerate at -20 (NEVER freeze)

-

gardnerella vaginalis culture media

-V agar (rarely cultured, so not really used)

syphilis serology

-nontreponemal: NDRL and PRP measure regain (nonspecific)

-treponemal: FTA-TP and TP-PA detect antibodies to T. pallidum

penicillinase producing nesseria gonorrhoeae (PPNG)

cannot treat with penicillin

-test for production of ß-lactamase in all isolates of N. gonorrhoeae

-nitrocefin/cefinase chromogenic substrate: when ß-lactam ringed is cleaved a color change occurs and tells you the isolate is drug resistant

-disk diffusion: penicillin disk is plated with isolate to look for sensitivity/ resistance

gonorrhea treatment

cefatriaxone

chlamydia treatment

azithromycin

dual therapy

ceratriaxone and azithromycin are given together to treat chlamydia or gonorrhea to make sure that a coinfection is treated

vulvovaginal candidiasis (vaginitis)

overgrowth of candida albicans in the vagina leading to thick discharge and itch

trichomoniasis

infiltration of trichomonas vaginalis parasite

-urogenital flagellate can be seen motile in the urine

phenotypic diagnostic tools

results can be altered by growth conditions

-biochemical characteristics

-immunologic assays

-MALDI-TOF

genotypic diagnostic tools

results are not altered by growth conditions

-DNA probes

-PCR

molecular biology advantages

-much faster than biochemical testing

-not affected by growth conditions that alter the phenotype of the bacteria

-highly sensitive and specific

-maldi-tof and others allow multiple samples to run at the same time

polymerase chain reaction (PCR)

-a method of producing copies of a target nucleic acid sequence

-allows you to analyze the amplified sequence to look for a specific gene or nucleic acid sequence in a sample

reverse trancriptase PCR (RT-PCR)

a single strand of RNA gives rise to many copies of complimentary DNA (cDNA)

-step 1: reverse transcriptase is used to convert the RNA strand and make a cDNA

-step 2: cDNA is amplified to make many copies

real time PCR (Q-PCR)

the accumulation of PCR amplicons is detected in real time as it happens, using fluorescent labels

-fluorescent labels are used to quantify the DNA and count how much is made in each round of amplification

amplicon

copies of target DNA sequence that are produced by PCR

PCR reaction components

-template DNA: intact DNA sequence of interest to be amplified

-DNA bases (dNTPs): dAT, dCTP, dTTP, dGTP

-DNA polymerase, Taq polymerase

-reaction buffers

-2 oligonucleotide priemrs

multiplex PCR

using more than 1 type of primer pair in a PCR in order to amplify multiple target DNA sequences at the same time

-*allows you to detect multiple pathogens at the same time in a sample*

-each primer is specific to a different pathogen

-primers have different annealing temperatures

-amplicons are able to be differentiated

nested PCR

PCR with increased sensitivity and specificty that uses 2 different primer sets

-1 primer set to amplify target DNA,

-2nd primer set to amplify the amplicons (copies) produced from the target DNA

MALDI-TOF MS (matrix assisted laser desorption/ionization-time of flight mass spectrometry)

-biomolecules in a specimen are vaporized into a gas, then ionized by a laser

-ions are separated based on molecular weight (mass to charge ratio / m/z ratio)

-ions with smaller m/z ratio are more charged and travel faster through the drift space to reach the detector

-ions with larger m/z ratio move more slowly

-a spectra of all the proteins in the sample is produced and compared to spectra of known organisms to identify the organism in the sample

mass to charge ratio (m/z)

in MALDI-TOF MS this determines how quickly the time of flight of an ionized particle occurs

normal biota of blood

NO normal biota, sterile fluid

-any bacteria isolated is significant

-normal biota of skin can contaminate a specimen: corynebacterium spp., CoNS, cutibacterium acnes, streptococcus spp., bacillus spp.

bacteremia

organisms that are present in the blood, not causing any symptoms

-transient, cleared out by immune system

septicemia

organisms that are present in the blood, causing symptoms and sickness

-immune system is not able to clear them out

endotoxin (LPS)

LPS from gram negative cell wall that is part of the organism

-acts as a toxin in the blood stream

exotoxins

toxins that are secreted from bacteria into their surroundings

-gram positive and gram negative bacteria

toxemia

presence of endotoxin and/or exotoxins in the blood stream

-when treating a gram negative blood stream infection, be careful that killing the bacteria too rapidly will cause rapid release of endotoxin that makes symptoms worse for a period of time

bacteremia transmission

-microbes penetrate through the epithelium or mucosal membranes (esp. a break in the skin)

-infection can stay localized or spread to other sites in the body

-bacteria spreads through lymphatics or blood stream, and around the body

-in rare cases infection can spread through the nerves

transient bacteremia sources

-spread of pathogen from an infected body site

-indwelling prosthetic devices

-dental/ surgical procedures

-IV drug abuse

septic shock continuum

-systemic inflammatory response syndrome (SIRS): immune system activates and produces cytokines that cause inflammation, fever/ hypothermia, tachycardia, tachypnea, and hypotension that leads to poor perfusion of organs

-severe sepsis: if not treated, bacteria is able to establish a full infection, and inflammation persists and becomes worse leading to organ failure

-septic shock: circulatory failure and hypotension occur leading to multiple organ failure, acute respiratory distress, DIC, tissue destruction/ necrosis and possibly death

SIRS lab findings

-abnormal WBCs: leukocytosis with band cells (left shift) or leukopenia

-organ failure: kidney, liver, etc.

-respiratory distress

-inflammatory and infection response active: CRP and procalcitonin elevated

predisposed to blood infections

-immunosuppressed: natural or induced

-underlying diseases: pregnancy, diabetes, existing kidney and lung disease, cancers

-age: very young or very old

-wounds, invasive procedures: e.g. blood catheter that introduces bacteria and provides a place for biofilm formation

-previous antibiotic therapy: decreased competition by normal biota

pericarditis

infection of the sac around the heart

-usually viral

-commonly caused by enteroviruses (e.g. Coxsackievirus)

myocarditis (carditis)

infection of the heart muscle

-usually viral

endocarditis

infection of the inner lining of the heart, and heart valves

-usually caused by bacterial vegetations (biofilms) on heart valves

phlebitis

infection of the vein

endarteritis

infection of the inner lining of the arteries

acute endocarditis

rapidly progressing infection in a previously healthy heart with no existing damage

-occurs in hours to days

-Staph aureus

subacute endocarditis

gradually progressive infection of a previously infected/ damaged heart

-occurs over the course of weeks

-viridans strep

-sometimes enterococci

common blood culture pathogens

-CoNS

-Staph aureus

-enterococcus

-candida

-enterobacteriaceae, P. aeruginosa, actinetobacter baumanii

infectious endocarditis risk factors

-heart damage: congenital defects, cardiac damage, prosthetic heart valves

-IV drug use

infectious endocarditis (causative agents)

-gram positive cocci: staphylococci, streptococci, enterococci

-usually normal biota

blood culture bottle regulations

-skin disinfection: alcohol then iodophor antiseptics at venipuncture sites

-volume of blood: 20 mL for adults (more blood= more chance of isolating organism), based on weight for children

-number of bottles: 3 bottles collected at separate times over 24 hours

-blood to broth ratio: 1:5-1:10

-timing: prior to antimicrobial therapy (maximum recovery of organism)

-incubation type: at least 1 aerobic and 1 anaerobic

-clots: prevent clots that trap bacteria and make it hard to recover

blood culture bottle contaminant

single positive for normal skin biota organism

-only 1 of the 3 culture bottles is positive

blood culture bottle transport

-transport to lab within 2 hours

-maintain at room temp or in 35 C incubator

nonculture blood infection diagnosis

performed after blood culture bottles come up positive and you know you have an organism, you would do this instead of subcultures

-molecular methodologies: NAAT, MALDI-TOF

-other: latex agglutination, etc. (not used much anymore)

acridine orange (blood culture)

if there is a really small amount of bacteria in the blood sample you would use this stain to detect the bacterial nucleic acids

-better than gram stain if organism is in small numbers

manual blood culture system

blood culture bottles are visually inspected to look for indicators of bacterial growth

-turbidity

-gas production bubbles

-hemolysis (dark/ black discoloration)

-colonies growing on RBC sediment in the bottle

-not really done anymore

automated blood culture system

blood culture bottles are put in a system that continuously monitors for bacterial growth, and alerts you when its positive

-usually monitors for increase in CO2 produced by bacteria

-once it's positive you work up the bacteria to ID it

blood culture workup

after the blood culture bottles show up positive begin the process of IDing the bacteria

-preliminary workup: gram stain, acridine orange stain, report positive culture and gram stain morphology to Dr. and document it

-organism ID: plate into subcultures, perform direct tests (bile solubility, latex agglutination, etc.), *molecular methods* (gene probes, accuprobe), AST using subculture , NOT directly from blood culture bottle

reporting blood culture results

-positive blood culture is a critical result and needs to be shared with physician immediately

-share salient information (e.g. gram stain) that will help to direct treatment

-negative cultures should be reported at 24, 48 hours and 5 days as a final negative

-document all communication in written or LIS report

sodium polyanethol sulfonate (SPS)

additive used in blood culture broths

-anticoagulant: prevents clotting that traps bacteria

-prevents natural immune factors in blood from killing bacteria

-neutralizes some antimicrobials (if given before culture was collected)

-down side: inhibits growth of N. gonorrhoeae, N. meningitidis and peptostreptococcus anaerobius, which are already difficult to culture

lysis centrifugation isolator blood culture system

-blood sample is collected, RBCs are lysed, sample is centrifuged to separate the pathogen from the plasma

-sediment from centrifugation is plated

-benefit: good for isolating mycobacteria, bartonella and fungi from blood

-negatives: labor intensive, easily contaminated

automated blood culture systems (brands)

-bactec: measures CO2 production via fluorometry

-DIFCO: measures pressure change due to gas production from bacterial metabolism

-BacT/Alert: measures increase in metabolic end products of bacteria via pH induced color change to yellow

-biomerieux vital: measures CO production and change in pH via fluorometry

arterial catheter tip culture

if blood catheter is suspected as causing a blood stream infection, it is removed and cultured

-collection: cleanse skin and remove catheter

-transport: place catheter in sterile container

-inoculation: use maki roll technique to roll the tip of the catheter along an agar plate and then incubate to see if organisms grow

pericardial fluid

fluid that is found in the sac surrounding the heart

-normally sterile fluid

special concern blood isolates

-hazardous isolates: francisella tularensis (tularemia) requires special precautions when working in the lab

-fastidious isolates: bacteria with no cell wall (e.g. mycoplasma) require media supplements like sucrose, mannitol and erythritol

-AIDS patients: suspect infections by opportunistic pathogens like fungi, mycobacteria and viruses

septicemia treatment

-antimicrobial agents: kill bacteria in the blood stream (careful w/ gram negs, influx of endotoxin possible)

-increase blood pressure: administer fluids and vasoconstrictive drugs

-oxygen

-hemodialysis: remove toxins in the blood