2. Organism
• Burkholderia pseudomallei
– the causative agent of melioidosis has been described
• almost a century ago and
• considerable progress in terms of
• diagnosis and treatment was achieved
• It is still recognized as
– “the unbeatable foe” - for several reasons, like
• under-recognition
• High case-fatality rate
• unacceptable relapse rate and
• a “time-bomb” effect for sero-positive individuals
• This bacterium still has many secrets left to be
revealed
3. Burkholderia pseudomallei
• Burkholderia pseudomallei is a
– facultative intracellular bacterial
saprophyte
– resides in soil
• especially in rice paddy field,
• stagnant water etc
– responsible for causing a
• potentially fatal and fulminating
infectious disease of
• human and animal called
melioidosis
4. Melioidosis
• It is a potentially fatal and fulminating infection that
– afflicts both human and animals
• Disease is Manifested as
– Community acquired pneumonia
– Multiple abscesses and
– Septicemia
• Disease of public health importance in
– many tropical and subtropical countries of the world
• The hyper endemic foci of this disease exist in
– northern Australia and South East Asian countries
– resides on either side of Wallace line
• the imaginary line which separates Australia from Asian countries
5. • Melioidosis is
• typically a tropical disease
• prevalent in areas between 200 North and 200 south of equator
•Total Melioidosis endemic country =52
Global Distribution
6. Global Distribution
• Since Bangladesh is located
– 230N of equator and
– cases of Melioidosis were sporadically detected, so
• Bangladesh is considered as a melioidosis endemic country
• In recent years
– melioidosis has been increasingly being reported from
– many new countries outside the known endemic regions
• It requires intense investigation to know, whether the organism is
– recently spread to these new areas or it is just being unmasked by
• better recognition
• increase awareness and
• Surveillance activities
7. Emergence vs. recognition
• The World Health Organization defines an emerging
disease as
– one that has appeared in a
• population for the first time, or
• that may have existed previously but is rapidly increasing
in incidence or geographic range
• One of the main problems in defining is, whether a
– disease is truly increasing in incidence (emerging) or
– just being increasingly recognized (pseudo-
emerging) is the
• accuracy, or rather inaccuracy of surveillance
8. Melioidosis as an emerging disease
• Melioidosis represents an
– excellent example of an emerging disease in two
respects:
– it is being
• reported increasingly in many countries; and
• it is being recognized for the first time in countries where it
has not previously been described
• However, the reported epidemiology reflects a
– complex interaction between genuine emergence and
increasing recognition (pseudo-emerging) as
• familiarity with the disease and its causative agent increases
amongst clinicians and microbiologists respectively, and
laboratory facilities and techniques improve
9. Emergence vs. recognition
• In the case of melioidosis, accurate diagnosis relies on
– the awareness of clinicians of the disease
– the availability of laboratory facilities that are sufficiently well
developed to confirm the diagnosis and finally
– familiarity of laboratory staff with the disease and its causative
organism
• Genuine increases may relate to
– environmental, climatic or behavioral changes, or
– to increasing numbers of those predisposed to the infection as
• treatment of underlying conditions such as diabetes mellitus improves
10. The contrast between Thailand and Burma
• In Thailand
– reported cases of melioidosis increase annually,
where as,
• in neighboring Burma
– the disease was first reported 100 years ago but
– now seldom if ever recognized, and
• few Burmese doctors had ever heard of melioidosis
– And now melioidosis cases are beginning to be
recognized once more
• exemplifies the
• influence of factors other than true incidence on the
perceptions of emergence
11. Global burden of melioidosis
• A 2016 modeling study estimated that there are
– ~165,000 cases of melioidosis in humans per year worldwide of which
– 89,000 (54%) are estimated to be fatal
• This study highlights that
– Under-diagnosis and under-reporting of melioidosis are a major issue
especially on the
• Indian subcontinent
– where 44% of cases were predicted to occur
• Predicted incidence for India, Indonesia and Bangladesh are
– ~52,500, ~20,000 and ~16,900 cases per year, respectively
12. Tip of the iceberg
• However, only ~1,300 cases
were reported
– per year worldwide since 2010,
– which is <1% of the estimated
annual incidence
• This indicates that
– The tip of the iceberg metaphor
has been commonly used to
describe the evidence for global
melioidosis distribution
• The highest percentage of
melioidosis cases is
– reported by Thailand where
– 3.6-5.5 persons per 100,000
acquire melioidosis
13. Neglected of the “neglected tropical disease”
• Melioidosis is a disease of the rural poor
• So, still considered as
– one of the most neglected tropical diseases (NTDs)
– so much neglected that
• it is not even included in the WHO list of NTDs
• However, it kills more people worldwide every year
– than diseases that are much known
• such as leptospirosis and dengue
14. Discovery
• The pioneering work of British pathologists
• Alfred Whitmore and his Indian Colleague
C. S. Krishnaswami
• first described melioidosis as a
• “glanders-like” disease among a
–morphia addict in Rangoon,
Burma, in 1911
• During their post-mortem investigations
– They discovered the bacilli
– and initially named as Whitmore bacilli
– The organism is
• very similar to Bacillus mallei (now known as
Burkholderia mallei)
• Causative agent of Glanders
15. Nomenclature
• Subsequent studies discovered that
– although similar, the organism differed from the
– etiological agent of glanders, Burkholderia mallei by virtue of
• a motility
• rapid growth and
• failure to invoke the Strauss reaction ( severe localized peritonitis
and orchitis) when inoculated into guinea pigs
• It can be assumed that a
– new, but closely related organism had been discovered
– the bacterium was then named Bacillus pseudomallei and
• the sufferings expressed as ‘Whitmore`s disease’ or
• often ‘pseudoglanders’
16. Nomenclature
• Due to the similarity with Pseudomonas group
,this bacterium was initially
– designated as Pseudomonas pseudomallei
• However, molecular characterization, involving
• 16S rRNA sequencing
• DNA: DNA homology
• cellular lipid and fatty acid composition
– revealed that
• it belongs to the class of β Proteobacterium,
whereas
• Pseudomonas belongs to ɣ Proteobacterium
17. Nomenclature
• The genus was then named after
– Walter Burkholder
– a credit to his founding works on the plant pathogen
Burkholderia cepacia
• Since this organism is
– distinct from pseudomonas species
• So, finally, termed as
– Burkholderia pseudomallei in 1992, which is the
– causative agent for Melioidosis
18. Melioidosis
• Initially the disease was named as
– Whitmore's disease after Captain Alfred Whitmore, and
• it is also termed as
– pseudo-glanders or
– morphia injector’s septicemia
• In 1913, in Malaysia
– a fatal ‘distemper-like’ outbreak occurred in laboratory animals at the Institute for
Medical Research
• Stanton and Fletcher identified
– Burkholderia pseudomallei as the causative agent , and
– published a paper on this out-break
19. Melioidosis
• Stanton and Fletcher
renamed the disease
to
– ‘melioidosis’ in
1921
• The term melioidosis was
named from the
– Greek word
• “melis” – distemper
of asses, and
• “Eidos” –
resemblance
20. Melioidosis was first recognised in Rangoon in 1911 by the British doctor Alfred Whitmore and
his assistant C. S. Krishnaswami, although the name of the disease was coined by Thomas
Stanton and William Fletcher. From the time when the aetiological organism was first identified,
it has been renamed many times: Bacterium (or Bacillus) whitmori, Malleomyces
pseudomallei, Loefflerella pseudomallei, Pfeifferella whitmori, Pseudomonas pseudomallei and,
finally, it was officially named Burkholderia pseudomallei in 1992. CDC, Centers for Disease
Control and Prevention.
21. Taxonomy
• Burkholderia is one of several genera
– belongs to the family Burkholderiaceae,
– in the order Burkholderiales
– of the Gram negative beta-proteobacterium
• The classification of Burkholderia
pseudomallei is based on rRNA/ DNA
homology and common culture characteristics
22. Taxonomic classification of Burkholderia
• The Scientific classification of the bacterium is as
following:
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Beta Proteobacteria
Order: Burkholderiales
Family: Burkholderiaceae
Genus: Burkholderia
Species: B. pseudomallei
23. Organism
– aerobic gram negative rod
• 1-2 µm long & 0.3-0.5µm
width
– Motile with
• 2 -4 polar flagella
– Staining is irregular with
bipolar (safety-pin)
appearance
• Due to presence of
intracellular deposits of poly-
β-hydroxybuterate
– Similar to Yersinia pestis
24. Ecologic adaptability
• Although the organism is a
– soil dwelling saprophyte, it can
– invade eukaryotic cell where it
– lives a parasitic life, and
– can even survive in phagocytic cells and cause
• Fulminant disease in immuno-compromised individual
• And latent infections in immuno-compitant person
– Facultative intracellular,
• just like, Mycobacterium tuberculosis
25. Genomics of B. pseudomallei
• This striking ecologic adaptability
may be due to its genome which
– represents one of the most
complex bacterial genome so far
sequenced
• It is one of the rare bacteria which
possess
– two chromosomes - 4.07 and 3.17
mega base pairs
– The larger chromosome is involved
predominantly with
• essential cellular functions such as
metabolism, growth and
replication
– the smaller contains genes involved
in
• survival and adaptation to
complex niches
Schematic diagrams of two chromosomes in the
B. pseudomallei strain
26. Stability
• Due to presence of this complex genome
– Burkholderia pseudomallei represent
– one of the most resilient organisms which are able to
– survive in different harsh, adverse environmental conditions
FOR PROLONG PERIOD (like Pseudomonas)
– including
• in prolonged nutrient deficiency (up to 10 yrs)
• in presence of antiseptic and detergent solutions
• in acidic environments at pH 4.5 for up to 70 days
• in dehydrated condition (soil water content <10% for
up to 70 day)
• in widely fluctuating temperature (24 0C to 42 0C) and even
• in tripled distilled water for more than 3 days
27. Growth Characteristics
Nutritionally the bacteria are
non-exacting and
nutritionally non-fastidious
Like Pseudomonas it
Grows on most traditionally used
bacteriological medium
The colonies vary from mucoid and
smooth to rough wrinkled
colonies become rough and wrinkled
on prolonged incubation
Grows well at both 370C and
420C
28. •Colony morphology varies between strains and between media
•Single isolate shows seven colony morphotypes from
• mucoid
•Smooth
• opaque to
• rough wrinkled
29. Diagnostic dilemma
The most striking feature is that
it produces mauve to pink color colony in MacConkey agar
resembling weak lactose fermentation
although the bacterium is oxidase positive and non-fermenter
The large wrinkled colonies look like environmental
contaminant
These create diagnostic dilemma and
most of the microbiologists become
confused with these colony characteristics and
consider them contaminant, and
Growths are often discarded as being of no clinical
significance
30. Natural habitats of Burkholderia pseudomallei
• B. pseudomallei is a bacterial saprophyte that
– resides in soil, especially in rice paddy field and
– stagnant surface water
• It is commonly found in the
– rhizosphere
• the layer of soil directly influenced by root secretions and soil microorganisms
• It was found to inhabit not only the rhizosphere and roots but
also
– aerial parts of specific grasses
• This raises questions about the
– potential spread of B. pseudomallei by grazing animals whose droppings
were found to be positive for these bacteria
31. HUMAN INFECTIONS OCCUR BY
• Skin penetration or Wound infection
•Contact with contaminated soil or water
•Ingestion
•Contaminated water
•Inhalation
•Dust from contaminated soil
•Rarely
•Person-to-person
•Animal-to-person
8
32. Virulence factors
Virulence
factor
Role in virulence Reference
Capsule Epithelial attachment, resistance to
complement mediated lysis
(Ahmed et al., 1999)
LPS Resistance to complement and defensin (Burtnick and Woods, 1999;
DeShazer et al., 1998)
Flagella Motility (DeShazer et al., 1998)
Pili Epithelial attachment; micro-colony formation (Brown et al., 2002; Essex-
Lopresti et al., 2005)
Quorum
sensing
Stationary phase gene regulation, including
secreted enzymes and oxidative stress protein
(Lumjiaktase et al., 2006; Song et
al., 2005)
TTSS3 Invasion and vacuolar escape (Stevens et al., 2003; Stevens et
al., 2002)
Morphotype
switching
Alteration of surface determinants for in vivo
phenotypic changes
(Chantratita et al., 2007)
33. •After infecting human, the organism is able to
• invade, survive and replicate within both phagocytic and non-phagocytic cells
•Burkholderia pseudomallei secretes
• N-acyl-homoserine lactones (AHL)
•which are signaling molecules involved in the
• quorum sensing machinery that is used to
• coordinate attacks against the host environment and biofilm formation
34. Epithelial attachment and cell invasion
• Toll-like receptors (TLRs) located on cell surfaces
– recognize pathogen-associated molecular patterns (PAMP
i.e., such as LPS and flagella) and
• mediating nuclear factor-κB (NF-κB)-induced activation of the
immune response
• releasing pro-inflammatory cytokines IL-1β and IL-18
• Cell entry is aided by
– flagella
– lipopolysaccharide (LPS)
– type IV pili and
– adhesins BoaA and BoaB
• B. pseudomallei then quickly escapes the vesicle by lysing the
membrane using T3SS, T6SS and T2SS
35. Immunopathogenesis
N Engl J Med 2012;367:1035-44
intracellular- invasion, survival
and replication in both phagocytic &
Non-phagocytic cells
Escaping endocytic vesicles
and Breakout into cytoplasm
& replicate
OR
infect other cells through
actin based membrane
protrusions which help in
spreading infection
36. Type III secretion system
• The type III secretion system (TTSS)
– comprises a molecular syringe (a structure made
of a filamentous needle to translocate effector
proteins into the surrounding milieu or cells)
– that is deployed on close contact with host cells
• TTSS plays an important role in the
– bacterial invasion as well as
• escaping from endocytic vesicles into host cell
cytoplasm
37. Intracellular survival and replication
• Once free in the cytoplasm
– B. pseudomallei are able to survive and
• replicate intra-cellularly and also cause either
• cell death and
• cell-to-cell spreading by
– formation of actin-based membrane protrusions and
– move via continuous polymerization of host cell actin at polar ends
– thereby facilitating
• spread to neighboring cells
• cell fusion and
• multinuclear giant cell (MNGC) formation
– T6SS and the type IV secretion system (VgrG-5) are essential to this process
38. Intercellular and Secondary spread
• As well as direct cell-to-cell spread
• B. pseudomallei can also
– spread to the bloodstream, causing sepsis, and
– infect antigen-presenting cells, which then can
• transport the bacteria to the lymphatic system and
• contribute to dissemination of infection to
• secondary sites
• However, the exact mechanism of secondary spreading
remains elusive
• Bacteria also remain viable in dendritic cells
– inducing maturation and trafficking to secondary lymphoid
organs
39. Latent or persistent infection
• In immunocompromise individual
– the organism causes acute manifestation of
melioidosis, whereas in
• immunocompetent person the
– bacteria remain quiescent and cause latent
infection
• Similar to, Mycobacterium tuberculosis infection
40. Latent or persistent infection
• B. pseudomallei can remain latent for extended
periods before
– immunosuppression or other host stress responses
• reactivate bacterial proliferation and melioidosis develops
• Reported latency periods have ranged from 19 years to
29 years
• indicating that B. pseudomallei can enter a
• dormant state and evade immune surveillance
• Neither the site (tissue or sub-cellular level) of
latency, nor
– the mechanisms by which B. pseudomallei remains
undetected are clear
41. Latent or persistent infection
• By contrast, high antibody titers detected in
patients
– years after an episode of acute melioidosis suggest
• continuous exposure or
• covert sequestration
– i. e., bacteria hiding in cryptic sites with
– down regulation of products
– B. pseudomallei has been found within the nucleus
• which could potentially act as a
– persistence site for later recrudescence
42. Vietnam Time bomb
• After many years
– if the person becomes
• immunocompromised or developed
• Co-morbidities like diabetes, chronic
renal failure
• the organism become reactivated
and acts like time bomb
Incubation may be as short
as 2 – 3 days, or
May remain latent for years
Reactivation occurs long time
after exposure (after 62 years)
43. Risk factors
• The most common risk factor to
melioidosis is
– Diabetes mellitus, which is present in
• >50% of all patients with melioidosis
worldwide
• Individuals with diabetes mellitus have a
12-fold higher risk of melioidosis
44. Risk factors
– Other known risk factors include
• exposure to soil or water (especially during the rainy season)
• male sex (probably because of a greater risk of environmental
exposure)
• age of >45 years
• excess alcohol consumption and liver disease
• chronic lung disease
• chronic kidney disease and
• thalassaemia (which probably causes neutrophil dysfunction
due to iron overload)
– Prolonged steroid use and immunosuppression can
also predispose individuals to infection
45. Clinical manifestations
• B. pseudomallei infection has
– protean clinical manifestations, and severity varies
from an
• acute fulminant septic illness to a
• chronic infection
– Chronic infection manifest as the presence of symptoms for >2
months
– accounting for 11% of all cases that may mimic cancer or
tuberculosis
• Sub-acute disease involves
– prolong febrile illness involving multiple abscesses and
– B. pseudomallei can be detected in bodily tissues and secretions,
including blood, urine, and pus
46. Clinical manifestations
• In patients with melioidosis
• the clinical presentation, severity and outcome are
affected by the
– presence or absence of host risk factors
– the route of infection
– bacterial load and
– the putative variation in virulence of B. pseudomallei
strains
• as well as the
– presence or absence of specific non-ubiquitous B.
pseudomallei virulence genes
47. Mimicker of maladies
• Clinical diagnosis is often difficult, as the spectrum
– varies widely between benign and localized abscess to
– severe community acquired pneumonia to
– Fatal septicemia
• For this reason, many authors have rightly referred
to
– this disease as “the remarkable imitator” and the
– “mimicker of maladies”
• since the disease can mimic, and produce similar symptoms
caused by
– pyogenic bacterial infections
– gram negative sepsis or
– tuberculosis
48. Chronic Latent Melioidosis
• Chronic melioidosis is an
– indolent disease capable of mimicking a
– wide variety of other diseases, particularly tuberculosis
• Mortality from chronic melioidosis is low
– provided appropriate therapy is initiated promptly
• Latent melioidosis is an entity that is
– postulated to exist, as
• active disease may appear many years after exposure
• Currently, there is
– neither means to diagnose latent melioidosis, nor a
– recommended antimicrobial treatment for
• individuals who are suspected to have it
49. Recurrent melioidosis
• Recurrent melioidosis may be due to
– relapse or re-infection
– Severity of the primary infection and
– duration and type of oral antimicrobial treatment are
the
• most important risk factors for relapse
• The management of relapse and re-infection are
– broadly similar, but
– distinguishing between the two has
• widely differing implications for disease control and
prevention
50. Species Affected
• Severe disease in sheep, goats
• Pigs (chronic form)
• Occasional infection
– Cattle, horses, dogs, cats, buffalo
– Monkeys, rodents, camels, alpacas
– Birds, tropical fish
• Incubation period
– Variable, days to years
52. Other Species
• Horses and cattle
– Neurologic signs
– Respiratory disease
• Dogs (rare)
– Dermal abscesses,
epididymitis, lameness,
leg swelling
• Rodents
– Very susceptible
53. Biological War weapon
• The US Centers for Disease Control and
Prevention (CDC) have classified
– B. pseudomallei as tier 1 select agents
• because of their biothreat potential
– tier 1 select agents present
» “the greatest risk of deliberate misuse with the
most significant potential for mass casualties or
devastating effect to the economy, critical
infrastructure; or public confidence”
54. Safety consideration
• B. pseudomallei is considered as a
– highly virulent organism
– responsible for causing potentially
life threatening disease and is
– classified as category B bioterrorism
agents by CDC
• So they require the use of
– biological safety level BSL-2 with
BSL-3 precautions for all
manipulations of cultures
– All patient specimens and culture
isolates should be handled while
wearing gloves and PPE in a BSC
55. Identification
• Acute infection require immediate treatment with
– Multiple antibiotics for prolong period
• Accurate and rapid identification of etiologic agents
is essential to
– Provide timely therapeutic intervention and to
– Prevent fatal outcome of the disease
59. Isolation of organism
• Culture remains the diagnostic “gold standard”
• B. pseudomallei grows well on
– most routine laboratory media, like
• Blood agar/ MacConkey agar when
– Isolated from sterile sites
• Selective media, like
– Ashdown’s media
– is required when specimens are obtained from non-sterile sites
• Selective Enrichment broth, like
– Ashdown’s enrichment broth
– is required when Number of organism is very low in non-sterile sites
– Especially in environmental sample, like soil
60. Growth in Blood Agar Media
• On Blood agar, colonies appear
– Small, smooth
– Creamy-white to grey , or
– Yellow-orange
– Non- hemolytic
– Slight metallic sheen,
– becoming dry and wrinkled in old
culture
• Colonies produce a
characteristic
– Sweet, musty, earthy odor
• However, sniffing is not
recommended for identification
purpose
61. Growth in MacConkey agar
– Produce round, mucoid
0.5-2 mm sized colony
– resemble a weak
lactose fermenter,
mauve color colony in
24 h
– Usually flat, may
develop metallic shin
– Produce dry, wrinkled
colony with further
incubation
62. Growth in Ashdown’s media
• Organism typically
appears as
– Small, glistening
purple colonies
after 24 h
– Changing to rough,
wrinkled colonies
with metallic shin
by 48 h
• Colonies produce a
characteristic
– Sweet, musty odor
63. Presumptive Identification
• Organisms isolated from culture media will be
Identified by
– Observing by colony morphology
– Gram staining
– Biochemical tests
– Antibiotic sensitivity tests (Diagnostic disc), and
64. GOLD STANDARD:
Isolation of B.
pseudomallei from
body fluids/
environmental
specimen
Metallic sheen with
dry and wrinkled colony
Colony morphology
65. Microscopy
• Gram staining reveals
– Gram-negative bacillus
with
– Uneven or bipolar staining
• Commonly known as a
‘safety pin appearance’
due to
• presence of poly-β-
hydroxybuterate, used for
carbon storage
• The bacilli are
– 0.8 to 1.5 µm in size
– Slender and vacuolated
with rounded ends
70. Phenotypic confirmation
• B. pseudomallei -specific
antigen detection methods
– Using monoclonal antibody
against 200kDa
exopolysaccharide, having
– 100% sensitivity and specificity
• Useful for rapid identification
of B. pseudomallei from
culture isolates
– Developed by
• Melioidosis research Centre
Khon Kaen, Thailand
71. Automated phenotypic identification systems
• Confirmation of identity is usually done
by
–Automated or semi-automated methods,
such as
• bioMeriux API 20 NE and
• Vitek systems
72. ENVIRONMENTAL SPECIMEN :
Soil
• Natural soil harbors a diverse bacterial and
fungal community
• Number of B. pseudomallei in soil is also
very low
• Hinders successful
– isolation of organism using non-selective
media
• This problem can be overcome by using
– Selective enrichment culture to increase the
number of isolates
– Highly selective media for selective growth
of B. pseudomallei, and
73. Processing of Soil sample for isolation and identification
of B. pseudomallei
Soil
20gm
3 ml Sup Cultured in 9
ml Enrichment Broth
Culture of enriched sup in
selective medium at 370C
Suspected colonies
cultured in MacConkey`s
medium at 420C for 24 to
48 hrs
Colonies positive at
420C with typical
morphology were
selected
Biochemical tests: Gram
stain, Oxidase, TSI, Citrate,
Urease, O/F sugar,
Raminoiglycoside & Colistin
Arabinose
Phenotypically B.
pseudomallei –
Confirmed by specific
Monoclonal anti-sera
48 hrs
20 g +60 ml dH2O
Mixed and vortexed
74. Rapid Diagnostic Tests
• Crucial for improving the outcome of melioidosis
• A lateral flow immunoassay (LFI; InBios, USA)
– developed to detect capsular polysaccharide (CPS) specific to B. pseudomallei
• Sensitivity varies with type of specimen
– 33% when tested with serum
– 47% when tested with pus
– 87% when tested with urine
– 99% when tested with turbid blood culture bottles
• Not yet commercially available
75. Molecular Diagnostics
• Advent of different molecular techniques have aided
in
– Decreasing the time required to confirm diagnosis for
infectious diseases
• High sensitivity is due to the ability to amplify
– A low copy number of DNA in the specimen
• High specificity is based on the
– Unique DNA sequence of the marker used
76. Genotypic detection:
• Variety of molecular detection
methods have developed
– Conventional PCR
– Real-Time PCR
– Loop Mediated Isothermal Amplification
(LAMP)
• Compared to conventional PCR
– Real-time PCR has added advantage of
automation with a
• Marked reduction in carryover
contamination
• Post-PCR manipulation also avoided
• Sequencing of conserved regions
77. Genotypic detection
• Variety of molecular detection methods have
developed
– Conventional PCR
– Real-Time PCR
– Loop Mediated Isothermal Amplification (LAMP)
• Compared to conventional PCR
– Real-time PCR has added advantage of automation with a
• Marked reduction in carryover contamination
• Post-PCR manipulation also avoided
• Sequencing of conserved regions
Confirmatory test
78. Target gene of B.
pseudomallei
• 16S Rrna
• flagellin (fliC)
• Ribosomal protein subunit S21 (rpsU)
• Type III secretion systems (TTS1 and
TTS2)
Confirmatory test
79. Loop Mediated Isothermal Amplification (LAMP)
• An in-house LAMP assay has been developed
– using a novel set of primers
• The in-house LAMP takes about
– 60 minutes for completion of reaction
• This molecular technique is able to detect
– 0.78 pg of B. pseudomallei DNA
• It can also detect the organism
– directly from body fluid, such as urine and pus, without nucleic acid extraction
81. DNA Extraction
Master mix
preparation
for LAMP
reactions
Master mix
preparation
for LAMP
reactions
without DNA
extraction
Heat Block
Left out pus
sample
from Melioidosis
case
LAMP from direct pus
sample
82. DIAGNOSTIC TESTS OF MELIOIDOSIS THAT CAN
BE INTRODUCED IN FUTURE
• MALDI-TOF-MS
• LFA-RPA
• Metabolomic profiling for identification of
novel biomarkers
83. Matrix-assisted laser desorption ionization-time of flight –mass
spectrometry (MALDI-TOF-MS)
• A revolutionary technique for
pathogen identification
• Yields
– rapid, accurate, and highly
reproducible results
– At a low price than any other
methods
• Methodology is easy with
– Minimum quantity of bacteria
required, and
– Results available within minutes
• Several studies addressed the
potential of MALDI-TOF MS
– All using the Bruker MALDI
Biotyper system
84. • Isothermal recombinase polymerase amplification combined
with lateral flow dipstick (LF-RPA) assay
• Result can be easy visualized in 30 minutes
• Limit of detection (LOD) as low as 20 femtogram (fg)
• Potentiality of early accurate diagnosis of melioidosis at point of
care or in-field use.
Lateral Flow Recombinase Polymerase
Amplification Assay (LF-RPA)
85. Metabolomic profiling for identification of
novel biomarkers
• The word metabolome appears to
be a
– blending of the words " metabolites "
and “chromosome”
• It implies that
– metabolites are encoded by genes or
– act on genes and gene products
• Metabolomics is the study of
chemical processes
– involving metabolites
– the small molecule substrates
intermediates and products of
metabolism
– The metabolic profiles from different
cells or systems can be used to
identify potential novel biomarkers
specific to these systems
86. Minimum identification criteria for diagnosis
• A simple set of criteria can be used in
laboratories where melioidosis is endemic
• These criteria includes identification by
observing
– Colony morphology in routine laboratory /
selective media
• Mostly rugose, mauve colonies with metallic sheen
– particularly after 48h
– growth at both 370C and 420C
– Gram stain
• Small gram negative rods with bipolar staining
– Oxidase positivity
– Resistance to colistin and aminoglycoside, and
– Susceptibility to augmentin (B. cepacia are
resistant)
