Burkholderia pseudomallei review including laboratory aspects

Guest posting: Dr Ian Marr, Microbiology Registrar, Pathology North.

Melioidosis is a cause of severe pneumonia, bacteraemia, urosepsis, prostatic abscess, CNS infection, pyogenic myositis and liver and spleen abscesses. It is found in PNG especially around the Balimo region – see Warner, Currie et al 2018 : Melioidosis in Papua New Guinea and Oceania- an important short read.

Melioidosis is caused by Burkholderia pseudomallei,  a Gram-negative rod, straight or slightly curved, with bipolar staining, or safety pin appearance. It is highly motile when viewed under phase contrast microscopy.

It is intrinsically resistant to colistin and gentamicin and therefore can be isolated on selective media containing these agents.

The best media for this is Ashdowns. This enables the other organisms to be suppressed and not overgrow Burkholderia (needed when isolates are from non-sterile sites).

Ashdown’s media contains:

  • Trypticase soy agar
  • Crystal violet – inhibits Gram positive organisms
  • Gentamicin – inhibits many Gram negative organisms (except B. pseudomallei)
  • Neutral red – absorbed by B. pseudomallei to differentiate from other bacteria
  • 4% glycerol – required by some B. pseudomallei strains for growth
  • Look for the characteristic flat wrinkled purple colonies by 48h

Other features include:

  • Strict aerobe, non lactose fermenter on MAC agar
  • Earthy odour (but important NOT to smell plates as this can cause laboratory acquired infections)
  • Catalase +ve, oxidase +ve
  • MALDI – there are limitations to using (MS) mass spectrophotometry currently. Often times it is mis-identified unless you have access to the security sensitive database on the Bruker, or you have your own curated library for this organism.
  • Can be misidentified by Vitek 2 (called Bcepacia, or thialandensis for instance)
  • API-E and NE can also mis-identify this organism
  • Australia sites where there is a lot of this organism use PCR targets (type III secretion system)
  • NEW* – Lateral flow assay – developed by InBios
    • 99% sensitive and 100% specific on positive BC samples
    • Place a drop of blood in with extraction mixture for 2-3min and then apply this solution to the lateral flow device. See this paper for recent trial in Laos.

Issues with handling:

  • RG2 (Risk Group 2) organism; laboratory-acquired infection reported so DO NOT USE AEROSOLISING PROCEDURES
  • Work in a biological safety cabinet; use appropriate PPE; avoid sniffing plates
  • Bactrim prophylaxis (3 weeks) for high-risk exposures; monitor for symptoms & seroconversion

Treatment / Antibiotic susceptibility 

  • High mortality – especially when immunosuppressed, diabetic, renal failure.
  • Intrinsic resistance to: penicillin, aminoglycosides, macrolides, colistin
  • Usually susceptible to: ceftazidime, meropenem, TMP-SMX-> augmentin and doxycycline used as second line agents
  • AST by disc diffusion is unreliable
  • E-test to determine MIC is necessary: TMP-SMX, meropenem, ceftazidime, doxycycline
  • Treatment: generally, 14-28 days IV ceftazidime, then >=3 months of oral eradication (TMP-SMX) depending on site of infection – See Royal Darwin Treatment 2014 guidelines.
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Virology overview

Consider specific diagnostic approaches to each agent, including where relevant, serological testing, antigen detection and nucleic acid amplification methods.  Also know what is locally in use in your laboratory.

Immunisation – need to know about the vaccines that are available – relevant for HPV, HBV, Influenza, Dengue, JEV, Polio, HAV, HEV, MMR, Rotavirus.

Particular attention required for those agents underlined. This is not an exhaustive listing but includes all viruses of current clinical relevance.

Reference

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Rabies review

Dr Ian Marr has produced a useful summary for our local ID & Micro tutes recently which is shared – Rabies_2018 Marr .

It includes reference to Australian bat lyssavirus which clearly may have an incidence outside of Australia (in bats and/or humans)! Serosurveillance of bat populations in the Philippines has suggested that lyssavirus infection of bats might be more widespread than previously thought.

For the purposes of post-grad UPNG pathology revision, I would suggest you consider these additional questions-

  1. What is the local epidemiology of rabies in SE Asia?  Especially focus on prevalence of cases within Indonesia.
  2. Read up the rabies vaccine advice provided by the Australian NHMRC Immunisation handbook.
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Leprosy – key resources

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Human Papilloma Virus – further resources

References

From Cepheid Xpert brochure:

cepheid performance

 

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NAMS Nepal presentations 2018 (R Martin)

Here are the four presentations given to National Academy of Medical Sciences, Bir Hospital Pathology and Medicine Residents over the week commencing 7/5/18  by Emeritus Professor Robert Martin, President of the Diagnostic Microbiology Development Program.

 

Elements of laboratory QMS (WHO)

References

 

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NAMS Nepal presentations 2018 (JK Ferguson)

Here are the three presentations given to National Academy of Medical Sciences, Bir Hospital Medical Residents over the week commencing 7/5/18. Thank you for attending and I hope they were useful. Please feel free to contact me via email if you have questions!  As mentioned, I do encourage you to enrol in the international online AMS MOOC course.    Best wishes, John

References

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How to read the cumulative antibiogram and its usefulness

Here are example antibiograms for Hunter New England (NSW). These are constructed by assessing the % susceptible for specific bugs and drugs, taking only the first isolate of a species per person per annum.  Where is it possible extrapolate susceptibility rather than test or we know a particular species is intrinsically susceptible, we enter “S”. Conversely, species that are intrinsically resistant to an antibiotic (e.g. Gram negatives and vancomycin) are marked as “R”.  In either situation, there is no rationale for actually testing that antibiotic against the species.

Continue reading

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Nocardia (part 2 topic)

Guest posting: Dr Syeda Navqi, Microbiology Registrar, Pathology North. 

Group: Aerobic actinomycetes

  • Gram positive bacteria that are usually filamentous and branched, commonly producing a fungus like mycelium that fragments into rod shaped or short coccoid form.
  • All grow better under aerobic than anaerobic conditions, a feature distinguishing them from most organisms in the genus Actinomyces.
  • The organisms containing mycolic acids in their cell walls (included in the genera Nocardia, Rhodococcus, Gordonia, Tsukamurella, and Corynebacterium) are rather closely related on the basis of molecular genetic studies.  c.f. Streptomyces, Actinomadura, Dermatophilus have no mycolic acid.
  • Nocardia is the most important genus as the most commonly isolated aerobic-actinomycete human pathogens.
  • There are approximately 87 validly named species included in Nocardia genus.

Identification

 Microscopic Morphology

  • Should be the initial step in organism identification
  • On direct Gram smears, organisms generally appear as very long, obviously branching, thin, and finely beaded Gram-positive rods
  • The modified acid-fast stain used on direct specimens as well as on colonial growth. Acid-fast cells will clearly be red; cells that stain purple or light pink may or may not be truly acid fast. The acid fast reaction has been reported to be most reliable when the test is performed with colonies after 1 to 4 weeks of growth.
  • Growth requirement and Medium Slow growing, require minimum 48-72 hrs before colonies appear, grow at room temp, incubate for 2-3 weeks.

Blood agar, chocolate agar, brain heart infusion agar, Sabouraud dextrose agar, and Lowenstein-Jensen medium support the growth of most aerobic actinomycetes; Buffered charcoal yeast extract agar (BCYE) is particularly useful for the recovery of Nocardia species. Specimens from sterile sites or concentrated sterile body fluids can be inoculated directly onto these media.

Specimens from respiratory sites, skin, and other potentially contaminated sites, such as mycetomas, should additionally be inoculated onto selective media, such as modified Thayer-Martin agar and selective BCYE (containing polymyxin B, anisomycin, and either vancomycin.)

Colonial morphology

  • Variable
  • Colony color may best be seen on the reverse when colonies are grown on translucent media (such as Sabouraud agar), as color may become obscured on the surface by the powdery aerial hyphae typically produced by members of this genu. Of the genera that are partially acid fast, only Nocardia species regularly produce aerial hyphae.
  • Chalky, matte or velvety, powdery irregular, wrinkled, or smooth; generally apparent  pink, orange, red, purple, gray, yellow, peach, or white on the reverse; smooth or granular; soluble brown or yellow pigments may be produced.
  • Nucleic acid amplification: molecular methods for the detection of Nocardia directly from clinical specimens are well developed
  • MALDI-TOF: some limitations to date

Susceptibility Testing

  • Basic biochemical testing may be paired with susceptibility testing to achieve preliminary identifications.
  • Some species or species complexes have predictable susceptibility patterns that may assist in isolate characterization.
  • These patterns should not be used exclusively as identification techniques, as many newly described species have not been tested for their antibiotic susceptibilities.

Epidemiology and clinical significance

  • Normal inhabitants of soil and water that are responsible for decomposition of plant material.
  • Facultative intracellular pathogen capable of growth in various human cells.
  • Infections can occur in both immunocompetent and immunocompromised hosts.
  • N. asteroides, N. brasiilensis and N. otitidiscavarium are the major cause of infections.
  • Infections generally result either from trauma-related introduction of the organism or, particularly in immunocompromised patients, from inhalation and the resulting establishment of a pulmonary focus.
  • Extrapulmonary disease  usually results from haematogenous spread from a pulmonary site; the brain is one of the most common secondary sites of infection.
  • Various Nocardia species implicated as the causal agents of keratitis and other ocular infections.
  • In 1988, a breakthrough in the clinically useful categorization of pathogenic nocardial isolates was provided by Wallace and his coworkers. They divided organisms phenotypically resembling N. asteroides into six different drug pattern types and one additional miscellaneous group. With more-recent molecular characterizations, numerous different species have been described within this set of organisms, which came to be known as the Nocardia asteroids complex. These include
  • Nocardia abscessus (drug pattern I),
  • Nocardia cyriacigeorgica (drug pattern VI),
  • farcinica (drug pattern V),
  • Nocardia nova (drugpattern III),
  • Nocardia wallacei (drug pattern IV), and isolates of drug pattern II.
  • Other new Nocardia species are continually being described, and undoubtedly,by current species definition criteria, many more will be described in the future.

Differentiation of Nocardia from other Aerobic actinomycetes

Genus Gram stain Modified acid fast Colonies morophology Growth in lysozyme Urea hydrolysis

 

Nocardia Branching filaments,beaded + Dry chalky, heaped or folded,yellow to grey white, pungent musty odour

Extensive aerial hyphae

+ +
Streptomyces same _ Similar to nocardia but do not fragment easily

Extensive aerial hyphae

_ +/-
Rhodococcus Coccoid to bacillary forms +/- Salmon colour with no musty odour

Minimal aerial hyphae

+/- +/-

 

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Recent update on TB diagnostic techniques – GenXpert MTB/RIF Ultra

Guest posting: Dr Ian Marr, Microbiology Registrar, Pathology NSW, Hunter

The GenXpert used in most LMIC has cartridges for detection of Mycobacterium tuberculosis (MTB) – GenXpert MTB/RIF. These contain primers and molecular beacon probes for the detection of both MTB and its resistance to the first line drug rifampicin. By looking for mutations in the rpoB gene (transcribes for RNA polymerase) it can determine if rifampicin (which inhibits RNA polymerase) will work and also if MTB is present.

What has changed?

There is a new GenXpert cartridge developed by Cepheid that has improved sensitivity for detecting Mycobacterium tuberculosis in clinical samples – MTB/RIF Ultra. While its sensitivity for detecting the rpoB mutation is approximately the same, there has been an increase in sensitivity for detecting the presence of MTB by addition of unique primers for IS6110 (an ‘insertion sequence’- basically a transposon that may have more than one copy in each genome).

Early in vitro validation reports indicated that the Ultra cartridge may be as sensitive as culture, however clinical samples while showing improved sensitivity, have not seen this. See Chakravorty et al., for further information on in vitro validation of the assay and to understand the changes in molecular primers and probes.

What does the clinical data say?

The two most interesting studies so far have been completed in CNS disease and pulmonary disease.

CNS disease

This study in Ugandan HIV positive persons was completed retrospectively on stored CSF samples. The Ultra cartridge showed a sensitivity of 70% (95% CI 47–87; 16 of 23 cases) for probable or definite tuberculous meningitis compared with 43% (23–66; 10/23) for the current cartridge. This compared to 43% (23–66; 10/23) for culture. While the study was of only small numbers it showed a significant increase from the previous GenXpert cartridge.

Pulmonary disease

This study was completed in 8 countries, analysing 1753 patients with TB symptoms found a variable sensitivity a little better than the current cartridges.

Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0).”

Does this mean that Rifampicin resistance also has an increase in sensitivity?

Unfortunately, despite re-engineering of the rpoB sloppy molecular beacons there was little if any increase in sensitivity of the rifampicin resistance component.

Is there any change in the report generated?

A change in the method of positive MTB detection now allows for a report of how much DNA was in the specimen. This is reported as either “trace”, “very low”, “low”, “medium”, or high”. Where there is only a small amount of IS6110 found no rpoB analysis will be reported.

Interestingly a number of false positives were also found in the ‘trace’ only reports in the pulmonary study above. Some of these were in patients who previously had MTB and had completed treatment. This may be because of DNA remnants left behind in these patients.

What does all this mean?

With an increase in sensitivity it is likely that this new cartridge will take over from the current MTB cartridge, and the reports generated will be different.

References (all are free full access)

 

Image credit: Wikipedia

 

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