Sunday, July 31, 2011

The End!

This truthfully has been a great semester, and a great learning experience. I really wish had more time to put into in, because I honestly think i have learned tremendously from all this material during this class. I honestly never thought I would even be able to look at a plate with 3 or 4 organisms and would be able to differentiate between all of them.
I enjoyed this class with everyone, hopefully someone has learned something new from my posts.
I really liked this idea of doing blogs, because I honestly had to search and think about new ideas and materials every time, and that expands my learning exponentially in the topic area. Even though sometimes it was hard for me to come up with something different for both the lecture and lab.
This has been a great experience, and hope to see you all next semester.
Good luck everyone on your finals!!!!!!

Sunday, July 24, 2011

CDC Guidance for Clinical Laboratories for conducting Influenza Testing

This past week our topic was the Laboratory Methods in Basic Virology. Influenza virus, especially the H1N1 strand, has caused an epidemic worldwide during the last few years. I will be discussing in this thread the CDC guidance for processing of influenza testing in the clinical laboratory.

Many different laboratory diagnostic tests can be used for detecting the presence of influenza viruses in respiratory specimens. These tests include direct antigen detection tests, virus isolation in cell culture, or detection of influenza-specific RNA by rRT-PCR methods.  
At this time, there are only two FDA approved assays for confirmation of novel influenza A(H1N1) virus infection, including the CDC rRT-PCR Swine Flu Panel assay. Confirmation of influenza A(H1N1) infection may be necessary for surveillance purposes and for special situations, such as immunocompromised patients or in pregnancy.

Rapid influenza diagnostic tests (RIDTs) are antigen detection tests that detect the influenza viral nucleoprotein antigen, and these assays are point of care tests.  Some of these tests, with high sensitivity and specificity, can detect and distinguish between influenza A and B viruses, but none can distinguish between the different subtypes.  The sensitivities of RIDTs to detect influenza B viruses are lower than for detection of influenza A viruses.  The sensitivities of RIDTs appear to be higher for specimens collected from children than specimens collected from adults.
Compared to RT-PCR, the sensitivity of RIDTs for detecting novel influenza A (H1N1) virus infections ranged from 10-70%. Therefore, a negative RIDT result does not rule out novel influenza A (H1N1) virus infection. The type of respiratory specimen (i.e., nasal vs. nasopharyngeal swab), quality of the specimen, time from illness onset to specimen collection, the age of the patient, time from specimen collection to testing, and the storage and processing of the specimen prior to testing, all are factors that contribute to the sensitivity of these tests.


Positive test result with RIDT indicates that the influenza virus infection is likely present in the specimen.  A negative rapid test result does not rule out influenza virus infection, and clinical presentation should always be taken into consideration. 
The specificity of RIDTs is generally high. However, especially during periods of low influenza activity (e.g. the very beginning of the season), however false positive results can occur.
The CDC recommends adding a statement when about the test limitations in the report of results so that the physician can decide how best to use the test for patient management.



"Example of a Statement to Accompany Rapid Influenza Diagnostic Test Results
RIDT result: Positive for Influenza Type A 
Note: This test can not distinguish influenza A virus subtypes.  For example, this test cannot distinguish influenza infections caused by novel influenza A viruses versus seasonal influenza A viruses.

RIDT result: Negative for Influenza A and B
Note:  The sensitivity of this assay has been shown to range between [10-70%*] for the detection of novel influenza A (H1N1) virus and between [20-100%*] for seasonal influenza viruses. A negative result does not exclude influenza virus infection. If influenza is circulating in your community, a diagnosis of influenza should be considered based on a patient’s clinical presentation and empiric antiviral treatment should be considered, if indicated.  If more conclusive testing is desired, follow-up confirmatory testing with either [viral culture or RT-PCR*] is warranted".


http://www.cdc.gov/h1n1flu/guidance/rapid_testing.htm

Monday, July 18, 2011

CLSI definitions for Succeptibility testing - Lab week 7

As everyone remembers, we  have done susceptibility testing this week during lab. 
The test is performed by applying a bacterial inoculum to the surface of a large Mueller-Hinton agar plate. After the plates have been inoculated  for 16–24 h at 35°C prior to determination of results, the susceptibility of each on the antimicrobials was graded according to the diameter of the inhibition zone, and then susceptibility was graded as either susceptible, intermediate and resistant. The diameter of the zone is related to the susceptibility of the isolate and to the diffusion rate of the drug through the agar medium.
According to the CLSI, those categories each has a certan clinical implication:
Susceptible

'The "susceptible" category implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the recommended dosage is used for the site of infection.
 
Resistant

'The "resistant" category implies that isolates are not inhibited by the usually achieveable concentrations of the agent with normal dosage schedules, and/or that demonstrate zone diameters that fall in the range where specific microbial resistance mechanisms (e.g. beta-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.
 
Intermediate

'The "intermediate" category includes isolates with antimicrobial MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates. The intermediate category implies clinical efficacy in body sites where the drugs are physiologically concentrated. This category also includes a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharamacotoxicity margins.
 
 

Saturday, July 16, 2011

Antibiotic Resistance news articles + CDC MASTER program - Week 7

Our main topic during this week lecture focused on antimicrobial resistance and the rise of resistant strains to the different drug regimen that we have available currently; from HIV, to MRSA, N. gonorrhea and others, this is becoming a major health concern and is causing a rise in patient morbidity and mortality, and a huge rise in healthcare cost.
I have taken advantage of this and looked up some articles in the news about the rise of antibiotic resistance and its impact on healthcare.

Furthermore, the CDC have initiated the Multi-level Antimicrobial Susceptibility Testing Resources (MASTER) program which is responsible of publishing resources, case studies, reference materials, news and others about such incidences. Please refer to the following link:

http://wwwn.cdc.gov/dls/master/archives.aspx?type=1


Below is an article published in USA today regarding this topic.

http://www.usatoday.com/news/health/2002-09-29-drugs-usat_x.htm

Antibiotic resistance on the rise
In the battle between bugs and drugs, the bugs are scoring some big wins.
Scientists here at an international meeting of the American Society for Microbiology warn that disease-causing microbes are becoming immune to a growing list of antibiotics, and new antibiotics and vaccines are barely keeping ahead of them.
"There are patients today in hospitals for whom there are no effective therapies," says Gary Doern, director of clinical microbiology at the University of Iowa, a panelist at a briefing here of the International Forum on Antibiotic Resistance.
Until recently, almost all drug-resistant bacteria were confined to hospitals, where a concentration of sick people and high antibiotic use contributed to the evolution of strains capable of evading antibiotic attack.
Now, Doern says, it's not uncommon for patients to come in the door carrying drug-resistant microbes that are circulating in the community. Among concerns highlighted by doctors at the Interscience Conference on Antimicrobial Agents and Chemotherapy:
  • Methicillin-resistant Staphylococcus aureus, or MRSA, accounts for more than half of hospital-acquired bloodstream infections caused by staph. In some cities, 31% of such infections outside the hospital are methicillin-resistant, and in nursing homes, 71% of staph infections are MRSA.
  • In some areas, about 50% of Campylobacter bacteria, the most common cause of diarrhea, are resistant to Cipro.
  • About a third of the germs most commonly responsible for severe pneumonia, Streptococcus pneumoniae, are resistant to penicillin in the USA, and about 25% are resistant to multiple drugs.
  • Resistance to fluoroquinolones, a newer class of drugs, also is on the rise. Researchers in Toronto reported Sunday that in 2001, 1.2% of the S. pneumoniae bacteria in Canada were resistant to levofloxacin, a fluoroquinolone introduced in 1996, compared with 0.9% in 2000 and 0.4% in 1999.
"We are living in a time where increasing drug resistance is frustrating treatment of common infections," says Roger Finch, professor of infectious diseases at England's University of Nottingham. "It is having an impact on hospitals and forcing us to use (antibiotics) we might have kept in reserve" to use only against the most persistent infections.
Antibiotics wipe out bacteria that are susceptible to them. But when used improperly, they can encourage the growth of bacteria that have adapted to them. And because bacteria can swap genes under the right conditions, drug-resistant germs can share their resistance mechanisms with other germs.
That is what is thought to have happened in the case of a Michigan woman reported this summer. She acquired the USA's first case of Staphylococcus aureus, a common bloodstream and skin infection, that was impervious to the antibiotic vancomycin, says Michael Rybak of Wayne State University in Detroit. Vancomycin has been known as the drug of last resort; it is used to treat infections that don't respond to other drugs. But in recent years, it has been used so often that intestinal bacteria called enterococci have become resistant.
The Michigan patient already was infected with vancomycin-resistant enterococci, and those microbes might have passed their drug-resistance mechanisms to the staph bacteria, creating what doctors call a "superbug."
As bacteria evolve resistance to more classes of antibiotics, drug companies are racing to create new ones. Rybak presented data Saturday showing that a drug being developed by Cubist Pharmaceuticals, daptomycin, was effective against vancomycin-resistant S. aureus.
Company officials say the drug has been tested in more than 2,500 patients, and they hope it will be reviewed for licensing within a year. In other studies, researchers reported that another new antibiotic, telithromycin, is effective in treating drug-resistant pneumonia.
Though new antibiotics are welcome, doctors say that unless patients and health care professionals learn to use them more carefully, the bugs will continue to outsmart the drugs.
Education strategies in hospitals and in communities, aimed at both doctors and patients, are achieving success in reducing overuse and misuse of antibiotics, researchers say.
"If we use antibiotics, some level of antibiotic resistance will emerge," Doern says. To slow that down, "we should use antibiotics only when needed, and, when needed, use the right one."

Monday, July 11, 2011

Thio broth- Lab

infections with Pasteurella multocida

http://f12network.com/craig/microbiology/0809S1/ch27_md/index_md.htm#Pasteurella%20Multocida


Pasteurella multocida is a bacteria belonging to the Pasteurellaceae family; it is a Gram-negative, non-motile coccobacillus that is often present in the upper respiratory tracts of many livestock, poultry, and domestic pets such as cats and dogs. 

According to the American Pet Association, approximately 10% of animal bites require medical attention; 1-2% eventually requires hospitalization. 
One of the most common symptoms of infection with P. multocida is respiratory infection manifesting as a nasal discharge, sneezing, congestion, conjunctivitis, and clogged tear ducts. Pasteurella infections also can cause abscesses under the skin that can be chronic, requiring surgery to correct. Some abscesses can cause central nervous symptoms like nystagmus, circling to one side, and wryneck or torticollis. 









http://emedicine.medscape.com/article/224920-overview

P. multocida  is a facultative anaerobe, which will grow at 37 degrees Celsius on blood or chocolate agar, but will not grow on MacConkey agar. Colony growth is characterized with a  "mousy" odor due to metabolic products. it is oxidase-positive and catalase-positive.

P. multocida virulence factor is due to a toxin, known as "Pasteurella multocida mitogenic toxin", which activates Rho GTPases, which then bind and hydrolyze GTP, and are important in actin stress fiber formation. This eventually leads to cytoskeletal reorganisation. 

Most Pasteurella isolates are susceptible to oral antimicrobial treatment with  one of the following drugs, or in combination: amoxicillin, amoxicillin/clavulanic acid, minocycline, fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin), and trimethoprim-sulfamethoxazole. 

Infections with P. multocida are mainly due to animal bites. Below are the organisms that can be found in different bites from animals, please refer to medscape article: “Animal Bites in Emergency Medicine.”

  • Dog bites
    • Staphylococcus species
    • Streptococcus species
    • Eikenella species
    • Pasteurella species
    • Proteus species
    • Klebsiella species
    • Haemophilus species
    • Enterobacter species
    • DF-2 or Capnocytophaga canimorsus
    • Bacteroides species
    • Moraxella species
    • Corynebacterium species
    • Neisseria species
    • Fusobacterium species
    • Prevotella species
    • Porphyromonas species
  • Cat bites
    • Pasteurella species
    • Actinomyces species
    • Propionibacterium species
    • Bacteroides species
    • Fusobacterium species
    • Clostridium species
    • Wolinella species
    • Peptostreptococcus species
    • Staphylococcus species
    • Streptococcus species
  • Herbivore bites
    • Actinobacillus lignieresii
    • Actinobacillus suis
    • Pasteurella multocida
    • Pasteurella caballi
    • Staphylococcus hyicus subsp hyicus
  • Swine bites
    • Pasteurella aerogenes
    • Pasteurella multocida
    • Bacteroides species
    • Proteus species
    • Actinobacillus suis
    • Streptococcus species
    • Flavobacterium species
    • Mycoplasma species
  • Rodent bites - Rat-bite fever
    • Streptobacillus moniliformis
    • Spirillum minus
  • Primates
    • Bacteroides species
    • Fusobacterium species
    • Eikenella corrodens
    • Streptococcus species
    • Enterococcus species
    • Staphylococcus species
    • Enterobacteriaceae
    • Simian herpes virus
  • Large reptiles (crocodiles, alligators)
    • Aeromonas hydrophila
    • Pseudomonas pseudomallei
    • Pseudomonas aeruginosa
    • Proteus species
    • Enterococcus species
    • Clostridium species


Other references:
http://www.copewithcytokines.org/cope.cgi?key=Pasteurella%20multocida%20toxin


 

Monday, July 4, 2011

BactiCard Neisseria - Lab

I am going to dedicate this section to discuss the main test done to identify the different Neisseria species, and that most of us had to do during lab this past week. 

The BactiCard Neisseria is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB) for Moraxella catarrhalis, prolyl aminopeptidase (PRO) for Neisseria gonorrhoeae, gamma-glutamyl aminopeptidase (GLUT) for Neisseria meningitidis, and ss-galactosidase (BGAL) for Neisseria lactamica

After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. The IB reaction is read for a blue-green color after 2, then the BGAL reaction is read at 15 min, also for blue-green color change. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. 

The following picture shows positive results on BactiCard Neisseria for M. catarrhalis, N. gonorrhoeae, N. meningitidis, and N. lactamica respectively.

Color Atlas of Medical Bacteriology By Luis M. De la Maza

In a study published in an article titled: "Evaluation of the BactiCard Neisseria for identification of pathogenic Neisseria species and Moraxella catarrhalis”, the BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates.