Every End is a New Beginning!

The semester is coming to a close and the infectious disease course is wrapping up. Our first semester of clinical microbiology involved a significant amount of memorization of biochemical reactions and unique identifiers for a wide range of organisms ranging from gram positive organisms, gram negative organisms, anaerobes, fungus, mycobacteria and parasites. Infectious disease gave us the opportunity to incorporate that knowledge into real world applications. I was given my unknowns at the hospital this week. I had 6 different sites (stool, urine, blood, knee aspiration, abscess, and sputum). I was lucky enough to get about 3 organisms for every site to identify! So far I have successfully identified all of them and I am just waiting to find out the results of the confirmatory test I am running for what I suspect is C. perfringens (boxcars and double zones). I love this stuff! I was offered a position in a hospital in Chattanooga, TN where I will be a generalist, but in the future I am looking forward to specializing in micro.

I hope you have enjoyed reading my blog. If I get an itch in the future I may continue to post information, but for now I will be studying for my ASCP exam! Wish me luck!

Ebolavirus

Ebola hemorrhagic fever (EHF) is a viral hemorrhagic fever and one of the most virulent viral diseases known to humankind. The Ebola virus was first identified in the western equatorial province of Sudan and in a nearby region of Zaire (now Democratic Republic of the Congo) in 1977 after significant epidemics in Nzara, southern Sudan and Yambuku, northern Zaire.  Ebolavirus is transmitted by direct contact with the blood, body fluids and tissues of infected persons, or by handling sick dead or infected animals. The virus interferes with the endothelial cells lining the interior surface of blood vessels and with coagulation. As the blood vessel walls become damaged and destroyed, the platelets are unable to coagulate, and patients succumb to hypovolemic shock. Here is a video with some general information on the virus.


http://www.youtube.com/watch?v=VeKQLA-WSnA

The Ebolavirus is a single stranded RNA virus with the characteristic threadlike structure of a filovirus. Before outbreaks are confirmed in areas of weak surveillance on the local or regional levels ebola is often mistaken for malaria, typhoid fever, dysentery, influenza, or various bacterial infections which may be endemic to the region. Diagnosing Ebola in an individual who has been infected only a few days is difficult because early symptoms, such as red eyes and a skin rash, are nonspecific to the virus and are seen in other patients with diseases that occur much more frequently. However, if a person has the constellation of symptoms described above, and infection with Ebola virus is suspected, the patient is isolated and the local state and health department are notified.  

There is no standard treatment for Ebola hemorrhagic fever and the mortality rate of the infection has been documented to be as high as 90%. Treatment is primarily supportive and includes minimizing invasive procedures, balancing electrolytes (since patients are frequently dehydrated), replacing lost coagulation factors to help stop bleeding, maintaining oxygen and blood levels, and treating any complicating infections. 

                                       

Antigen-capture enzyme-linked immunosorbent assay (ELISA) testing, IgM ELISA, polymerase chain reaction (PCR), and virus isolation can be used to diagnose a case of Ebola HF within a few days of the onset of symptoms. Persons tested later in the course of the disease or after recovery can be tested for IgM and IgG antibodies; the disease can also be diagnosed retrospectively in deceased patients by using immunohistochemistry testing, virus isolation, or PCR.


Ebolavirus is a public health concern in Africa and the Philippines. The lack of available treatment or vaccination, and a high mortality cause a significant amount of fear and anxiety in the general public during outbreaks. Here is a sobering video on statistical information concerning the ebolavirus.


http://www.youtube.com/watch?v=xQ-qIBB2U-k


The latest case of Ebolavirus infection was in May 2011. A 12-year-old girl in Uganda died from Ebola hemorrhagic fever. No further cases have been recorded.

AST and Tests for Resistance

This week in lab a number of tests were performed to determine the susceptibility and resistance of Enterobacter cloacae, an unknown gram-positive cocci, Streptococcus pneumoniae, MRSA, and M. catarrhalis. Antimicrobial susceptibility tests are done to determine what antimicrobial agents are effective against isolated pathogens in vitro. Results of antimicrobial susceptibility tests guide the clinician in initial and later treatment of the patient. They are also used for epidemiological purposes in detecting outbreaks of hospital acquired infections and in locating resistant strains in the community.

An agar disc diffusion susceptibility test was performed on Enterobacter cloacae. The surface of a Mueller Hinton agar plate was inoculated with a standardized suspension of the organism in three directions so that the entire surface was completely covered. Eight antibiotic disks were pressed on the surface of the plate and it was incubated overnight at 35 C. The antimicrobial agents diffuse from the disks into the medium in a circle. As the distance from the disk increases there is a logarithmic reduction in antibiotic concentration, creating a gradient of drug concentrations in the agar medium surrounding each disk. The bacteria inoculated on the surface that are not inhibited by the antibiotic grow flush with the disk and no zone of inhibition is evident. In areas where the concentration of drug is inhibitory no growth occurs.

An MIC of an unknown gram positive cocci was also performed. Various antimicrobial agents are diluted in Mueller-Hinton broth supplemented with calcium and magnesium and dried in the microwells. After inoculation and rehydration with a standardized suspension of organism and incubation at 35 C for a minimum of 16 hours, the MIC for the test organism is determined by observing the lowest concentration showing inhibition of growth.

An E test on Streptococcus pneumoniae was also performed. The E test (also known as the Gradinet Diffusion Method) is based on the same principle as the disk diffusion method. It is an in vitro method for quantitative antimicrobial susceptibility testing whereby a preformed antimicrobial gradient from a plastic-coated strip diffuses into an agar medium inoculated with the test organism. The MIC is read directly from a scale on the top of the strip at a point where the ellipse of organism growth inhibition intercepts the strip.

Selective and differential medium for qualitative direct detection of methicillin resistant Staphylococcus aureus (MRSA) was also determined using CHROMagar MRSA. The medium permits the direct detection and identification of MRSA through the incorporation of specific chromogenic substrates and cefoxitin. MRSA stains will grow in the presence of cefoxitin and produce mauve colored colonies resulting from hydrolysis of the chromogenic substrate. Additional selective agents are incorporated for the suppression of gram negative organisms, yeast and some gram-positive cocci. Bacteria other than MRSA may utilize other chromogenic substrates in the medium resulting in blue to blue/green colored colonies or if no chromogenic substrates are utilized, colonies appear white or colorless. 

A Nitrocefin Disk for beta-lactamase on M. cattarrhalis was also performed. Nitrocefin disks are used for the rapid detection of β-lactamase enzymes in isolated colonies of Neisseria gonorrhoeae, Moraxella catarrhalis, Staphylococcus spp., Haemophilus influenzae and anaerobic bacteria. A positive beta-lactamase result is recorded when the Nitrocefin Disk changes in color from its original yellow to orange or red. Most positive bacterial strains will produce a color change within 5 minutes. Some staphylococci, however, may take up to 60 minutes for a positive result. A positive beta-lactamase result predicts the following:

1. Resistance to penicillin, ampicillin and amoxicillin among Haemophilus spp., N. gonorrhoeae and M. catarrhalis.

2. Resistance to penicillin, as well as acylamino-, carboxy-, and uriedo-penicillins among staphylococci and enterococci.

A negative beta-lactamase result is recorded when the Nitrocef Disk™ remains yellow in color. A negative result does not rule out resistance due to other mechanisms.  

Below is a link to the University of Pennsylvania's Medical Center explanation of antimicrobial susceptibility testing. Click on the links within the site for more detailed information:


http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/amt.html

Antimicrobial Susceptibility Testing

Susceptibility testing involves testing pathogens against antimicrobial agents to determine if the bacteria express resistance to agents that could potentially be used in treatment. The microdilution method of measuring antimicrobial susceptibility is commonly employed in the clinical laboratory. The procedure begins by preparing a standard suspension of the organism and dispensing it into the seed tray. A specialized inoculator device with prongs dispenses 0.01 mL of inoculum into wells of microdilution trays containing various concentrations of antimicrobial agents. The plates are continuously monitored by an automated system.The instrument detects the growth in the wells by spectrophotometry (or fluorometric analysis) by comparing wells with antimicrobics to control wells without antimicrobics. 

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The antimicrobial agents that are chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery. Antimicrobials to which the organism is intrinsically resistant are routinely excluded from the test battery. Similarly, certain antimicrobials were specifically developed for use against particular organisms but not others and should be included in the appropriate battery. If resistance to a particular agent is common, the utility of the agent may be sufficiently limited so that routine testing is not warranted and only more potent antimicrobials are included in the test battery. Conversely, more potent agents may not need to be in the test battery is susceptibility to less potent agents is highly prevalent. 

Further considerations about antibiotics may be employed against particular organism groups. The most common individual testing batteries are considered for Enterobacteriaceae, P. aeruginosa, Acenitobacter spp, Staphylococcus spp, Enterococcus spp, Streptococcus spp, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. Below is a link to an example of specific information for susceptibility testing of S. pneumoniae.

http://www.cdc.gov/abcs/reports-findings/downloads/lab-methods.pdf

Pasteurella multocida

Pasteurella multocida is a nonmotile gram negative coccobacilli. The organism grows best
 at 37 C and exhibits both oxidative and fermentive metabolism. P. multocida produces grey, rough, viscous, non-hemolytic colonies on blood agar and no growth on MacConkey agar. They are also catalase and oxidase positive with a characteristic "mousy"odor.

Pasteurella species are commonly isolated pathogens in most animal bites, especially in dog- and cat-related injuries. These injuries can be aggressive, with skin manifestations typically appearing within 24 hours following a bite that exhibit a rapidly progressive soft-tissue inflammation.

If left untreated Pasteurella infections can lead to cellulitis and can spread to cause serious systemic infection. Most reported cases of Pasteurella infections  involve skin and soft tissue with occasional development of regional lymphadenopathy. Beyond skin and soft tissue, other sites of infection are uncommon and have been the subject of individual case reports or small case series (they include bone and joint infections, CNS infections, septicemia, endocarditis, respiratory tract infections and intra-abdominal infections).

For more information about P. multocida or for a quick reference for other organisms visit the following link:

http://www.amrita.ac.in/bioprojects/IndusMicroBio/Micro.php

Armadillos and Leprosy

Mycobacterium leprae is an acid fast Gram-positive bacterium, with a slow doubling time of 14 days. The slow doubling time is due to the restricted intake of nutrients through the pores in the large waxy walls. Mycobacteria, as genus, are typically found in the soil, water and in the air.Leprosy is very specified when it comes to infecting hosts. Its ideal conditions are around 33 degrees C, which is lower than most mammals. Mammals with lower temperatures are better hosts for leprosy. That is why only a few species are known to be carriers of M. leprae. This is also why in humans, leprosy tends to be found primarily at the peripheral nerves. Hands and feet tend to be cooler than the core body temp
erature, providing a more habitable environment for M. leprae.



Mycobacterium leprae is an acid fast Gram-positive bacterium, with a slow doubling time of 14 days. The slow doubling time is due to the restricted intake of nutrients through the pores in the large waxy walls. Mycobacteria, as genus, are typically found in the soil, water and in the air.Leprosy is very specified when it comes to infecting hosts. Its ideal conditions are around 33 degrees C, which is lower than most mammals. Mammals with lower temperatures are better hosts for leprosy. That is why only a few species are known to be carriers of M. leprae. This is also why in humans, leprosy tends to be found primarily at the peripheral nerves. Hands and feet tend to be cooler than the core body temperature, providing a more habitable environment for M. leprae.


http://www.ksat.com/video/27710534/index.html

Shigellosis

Shigella spp. are nonmotile, non-spore forming gram negative bacilli that infect the intestinal tract of humans causing bacterial dysentery.  Shigella spp. are transmitted from person to person via fecal-oral route either by direct physical contact, or indirectly by ingestion of food or water contaminated with human feces. Symptoms of Shigellosis include diarrhea, bloody stool, abdominal cramps, and fever. Typically the first sign of Shigellosis is profuse watery diarrhea due to the presence of enterotoxin. The blood and pus in the stool develop as a result of the invasion of Shigella spp into the mucosa of the colon. A variety of adhesins enable the Shigella spp. to come into contact with the intestinal mucosal cells. Once across the mucosa, Shigella spp. use invasins to enter the epithelial cells where they escape from the vacuole into the cytoplasm and multiply. They survive phagocytosis by inducing apoptosis in macrophages.  Here is a video from the CDC with some general information on Shigella:

http://www.youtube.com/watch?v=kufdtZzSn70

Shigella spp. are identified in the clinical microbiology laboratory by growth on MAC and XLD/HE agar, as well as biochemical tests such as KIA, LIA and Urea. Shigella does not ferment lactose and appears clear on MAC agar. Hektoen enteric agar (HE) is a higly selective media that primarily recovers Shigella and Salmonella spp., inhibits common colon flora, and contains indicators to detect hydrogen sulfide production. Shigella appears green without black centers on HE agar b/c it does not produce hydrogen sulfide. When Shigella grows on Xyline-lysine deoxycholate agar it appears red or clear b/c it does not ferment any of these carbohydrates.

Shigella KIA results are Alk/A, LIA results are P/Y, and Urea testing is negative.

KIA- organism ferments glucose, organism does not ferment lactose (Alk/A)

LIA- Pink/Yellow

Urea- Negative

Additional testing may be performed to determine the grouping of Shigella spp. by agglutination of specimen with antisera fro Groups A (S. dysenteriae), B (S. flexneri), C (S. boydii), and the most common D (S. sonnei).

See you next time!

Legionnaire's Disease

Legionnaire's disease is a febrile pneumonic illness with numerous clinical presentations. Legionella pneumophila, the etiologic agent of Legoinaire's disease most commonly identified, is a faintly staining, thin, gram negative rod that requires media supplemented with iron and L-cysteine.

Legionellae are found primarily in aquatic habitats and can infect and multiply within some species of free living amoeba, protozoa, or within biofilms. Legionella infections are acquired exclusively from environmental sources. Inhalations of infectious aerosols is considered the primary means of transmission. Following infection the organisms are taken up by alveolar macrophages where they survive and replicate, evading the hosts immune system. This link provides an animation of L. pneumophila entry into the macrophage.

http://www.youtube.com/watch?v=dD4d_apLtl0&feature=related

Pneumonia is confirmed by chest x-ray or clinical diagnosis. The most commonly used laboratory test is the urine antigen test which detects Legionella bacteria in the urine specimen. A documented case of pneumonia and a positive urine test is diagnostic for legionnaire's disease. Diagnosis of legionnaire's disease can also be confirmed with isolation of Legionella pneumophila in culture from a lung biopsy, respiratory secretion, or from various other sites. In addition, paired sera that show an increase in antibody levels when drawn shortly after illness and several weeks following recovery can confirm diagnosis.

For you entertainment, I have added an additional link to a story about a suspected Legionnaire's outbreak at the playboy mansion. There is also a video link to an outbreak in a Miami hotel on the same page, in which there was one fatality.

http://abcnews.go.com/Health/legionnaires-outbreak-traced-back-playboy-mansion/story?id=12924405

Smoke and Mirrors

Day #1:

This week in lab we were given CSF samples that were plated on chocolate agar, SBA, and in thioglycolate broth. The source of the CSF was from a 19-year old male showing classic signs of meningitis. When I gram stained the CSF sample it was apparent that I had gram negative diplococci which led me to suspect Neisseria sp. The gram stain finding correlated well with the age and symptoms of my patient, considering Neisseria meningitidis is the most common cause of purulent meningitis in young adults. Unfortunately I was not able to make a definitive identification due to insufficient inoculation of the specimen used for the API NH...Lesson Learned!

This is what the API NH results looked like. Everything was negative! Ooops!

In addition we were given a urine sample plated on SBA, MAC and Chromagar. One day #1 I invested too much time on the significance of the turquoise colonies that appeared on my Chromagar. The organism that shows this colony morphology on Chromagar is Enterococcus.

But the confusion set in when I checked out my MAC agar...

It may be hard to tell by this picture but it appeared I had a NLF gram negative rod! Hmmmm, definitely wasn't the gram positive cocci Enterococcus. So after much confusion and and searching through flowcharts I set up an API 20E for definitive identification of my organism.

Day #2:

The next day when I arrived in lab I check out my MAC and Chromagar plates again. Things started to make more sense.

It was revealed that I had a slow WLF on MAC, which alleviated some of the confusion. And secondly, my colonies on Chromagar were metallic blue, suggestive of Citrobacter sp. It was starting to come together!

My suspicions were confirmed by the API 20E results. Phew! So, as Dr Greenup would say...it's time for reflection. My experience in lab this week taught me that when working with living organisms things may not always go by the book (or be what they seem). Identifying organisms is tricky. It takes patience, diligence, and experience!

Also I found a paper that summarizes clinical bacteriology quite well. It has some helpful flow charts that you may find useful when studying for your certification exam.

http://pmj.bmj.com/content/77/905/148.full

See you all next week!

Urinary Tract Infections

Hello everyone! The topic of discussion in yesterdays lecture was urinary tract infections. I will begin my post by summarizing the types of UTI's and then post a link with more detailed information.

Bacteriuria is defined as the presence of bacteria in the urine. The source of the bacteria is most likely associated with a UTI, which can be divided further into asymptomatic, pyelonephritis, urethritis, cystitis, and acute urethral syndrome.

Type of Bacteriuria	Site of Infection	Symptoms
Pyelonephritis	Upper UTI, kidney parenchyma, calyces, and pelvis	Vomiting, diarrhea, chills, increased heart rate, lower abdominal pain
Cystitis	Lower UTI, Bladder	Dysuria, increased frequency, suprapubic tenderness, hematuria
Urethritis	Lower UTI, Urethra, sexually transmitted	Frequency, dysuria
Acute Urethral Syndrome	Lower UTI, primarily infection of sexually active women	Frequency, dysuria, urgency

Predisposing factors that may lead to infection include compromised urine flow, alteration of pH and osmolarity due to hormones (pregnancy), high glucose concentrations, indwelling catheters, congential structural abnormalities, and immunosuppression. UTI's occurs most commonly in women due to shorter urethra, proximity of the urethra to the anus, and urethral contamination in sexual intercourse. 

Routes of infection that lead to Upper UTI's are divided into ascending routes and hematogenous, or descending routes. The ascending route is the most common. The bacteria migrate from the bladder to the upper urinary tract. The descending route occurs through the migration of organisms from the blood to the kidney.

The most common pathogens of UTI's include E. Coli, Klebsiella pneumoniae, S. saprophyticus, Poreus mirabilis, Pseudomonas aeriginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, and other Enterobacteriaceae species. The following table lists some common causes of UTI's and how they might be identified.

This link provide more detail about types of UTI's

http://www.atsu.edu/faculty/chamberlain/Website/lectures/lecture/uti.htm

Also, here is a link to a case study! You may have to copy and paste it into your address bar. Enjoy!

http://www.microrao.com/bact7.htm

Meningitis

On Thursday in lecture we discussed CSF examination in Infectious Disease.  Meningitis is characterized by inflammation of the membranes that cover the brain and spinal cord. It is divided into two categories: purulent and aseptic. Purulent meningitis is usually associated with bacterial infections and CSF examination revealing a large number of neutrophils. Aseptic meningitis is usually associated with viral infections and CSF examination revealing a large number of lymphocytes. The following table categorizes Purulent meningitis by age and condition and organisms commonly responsible for the infection.

Age or Condition	Organism
Neonates	S. agalactiae E. Coli Listeria GNB
< 5	N. meningitidis S. pneumoniae H. Influenzae (hib)
Young Adult	N. meningitidis
Adult	N. meningitidis S. pneumoniae
Elderly	S. pneumoniae Listeria S. aureus GNB
Aids Patients	Cryptococcus neoformans

Now we'll consider the following case history. See if you can identify the diagnosis and organism responsible for the infection in the following patient.

Case History:

A 38-year-old woman with no significant previous medical history came to the emergency department with a complaint of mild sore throat that had persisted for three days, accompanied by arthralgia, myalgia, and low-grade fever. The day before she had a severe headache with neck stiffness, nausea, and vomiting. Her white blood cell count was 21,800/mm3 with 67% polymorphonuclear leukocytes and 26% band cells. Platelet count was 200,000/mm3. Electrolyte levels were within normal range, and the glucose level was 131 mg/dL. A chest film showed no signs of pneumonia. A lumbar puncture revealed clear cerebrospinal fluid (CSF) with a glucose level of 88 mg/dL and a total protein level of 33 mg/dL and no cells. Approximately 12 hours later, she became acutely lethargic and a second spinal tap was performed. The CSF appeared cloudy and now contained 871 white blood cells, of which 93% were polymorphonuclear leukocytes; the glucose level was 1 mg/dL; the total protein level, 417 mg/dL. Gram stain revealed rare gram-negative diplococci.

In addition medium sized, smooth, gray to white colonies grew on chocolate agar. To discover the diagnosis and identification of the organism visit the following link...

http://www.emedmag.com/html/pre/fea/features/091500.asp

I hope you found this helpful! 

Streptococcus pneumoniae vs. Haemophilus influenzae

Before the battle begins I will brief you on my experience in lab this week...

It has been challenging trying to awaken my recollections of all of the information I learned in microbiology. Although it seems like yesterday, a year is just about enough time to forget all those little details. It's coming back to me now though. There were lots of bells ringing in lab this week.

It started yesterday when I received a blood culture bottle and case history for a 70 year old woman with multiple myeloma who was experiencing classic signs of a respiratory infection. I started off my investigation with a Gram Stain, but unfortunately nothing showed up. The only thing left to do was plate the blood culture out on chocolate agar and wait until the following day.

The following day and saw this!!!

Then I did a gram stain and saw this!!!

The presence of raised, umbilicate or flattened, gray colonies on the chocolate agar and gram positive lancet shaped diplococci on the gram stain directed my efforts to finding a confirmatory method for the identification of Streptococcus pneumoniae.  I performed a bile solubility test on two of the colonies. Bile solubility is performed to differentiate Streptococcus pneumoniae from other alpha-hemolytic species of streptococcus.  S. pneumoniae undergoes autolysis when exposed to bile salts by lowering the surface tension between the medium and the bacterial cell membrane. After the addition of bile salts my colony had diminished, which is a positive finding for bile solubility and the identification of S. pneumoniae. That is when I did some additional research on S. pneumoniae to determine the clinical significance and happened upon the battle between S. pneumoniae and Haemophilus influenzae.

Streptococcus pneumoniae vs. Haemophilus influenzae:


          IN VITRO: S. pneumoniae overpowers H. influenzae by attacking it with hydrogen peroxide.
        
          IN VIVO: H. influenzae is the only bacteria that survives when both are placed into the nasal cavity. That begs the question..."Why doesn't the hydrogen peroxide work for S. pneumoniae in vivo?" One of two hypotheses...The individuals immune system is triggered to attack S. pneumoniae when it attacks H. influenzae, or the presence of both species triggers a response that is not triggered by one species alone.

Interesting...for more information check out this link:
http://www.phschool.com/science/science_news/articles/bacterial_snitch.html

Welcome!

Hello and welcome to Infection Detection! For the next two months I will be taking an Infectious Disease course to fulfill curriculum requirements for the Clinical Laboratory Science program at UAB. As graduate students we are given an additional assignment in order to increase our understanding and knowledge of the subject. For the Infectious Disease course our assignment is to create a blog that catalogues topics covered in lecture and lab throughout the semester. Information will be posted twice a week and will include material covered in lecture and lab, something new that was learned, facts/data that was interesting, something that may have been unclear, and/or topics that I would like to know more about. 

As a future medical laboratory scientist it will be my responsibility to identify and evaluate bacteria, fungi, viruses, and parasites associated with infectious disease.  Journey with me throughout the educational process as I attain the knowledge and skills necessary to fulfill these responsibilities. 

Thank you,

Stephanie Reynolds