Combatting leptospirosis Increased awareness - niah.dld.go.thniah.dld.go.th/th/images/publicnews/033/lepto_file/... · 3 Amsterdam, The Netherlands Some approaches that have been

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Di ag nosis of leptospirosis a nd chara cte rization of Leptospira

Rudy Hartskeer l

Amsterda m, The Netherla nds www.kit.nl

Leptospirosis is globally emerging but still severely underestimated

>350,000 severe cases annually (ILS and WHO-LERG)

Reasons 1

•Mild to severe and uncharacteristic protean manifestations•Zoonosis with a complex and dynamic epidemiology

•Falls into (or rather between) several categories: zoonosis,

vector/rodent-borne, water-borne, food-borne, poverty, occupational disease

Reasons 2

•Difficult diagnosis (not recognised)•Cases not reported (no notification)

•Unawareness (Ignored)


Combatting leptospirosis

Increased awareness

Prevention and contro l measures (tay lor made)


Increased awareness

•Improved diagnosis; Diagnostic tests

•Notification and surve il lance

•Education, courses, meetings•International Leptospirosis Society (ILS)


Conventional testsConventional tests: Isolation, DFM, MAT, ELISA, IFAT,

IHA, MSAT etc.

Many drawbacks: slow, unrel iable, low detection

threshold, difficult to standardize, requi re wel l-trained personne l and/or expensive media/equipment


Rapid diagnosis: point-of-care tests•Easy•Simple•Reliable•Quick•Stable•Affordable

Negative Positive

Latex agglutination1 min

Lateral flow test10-15 min

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LEPTO lateral flow assay compared with MAT and ELISAin the Netherlands

<10 DPO >10 DPO

Sens % Spec % Sens % Spec %

MAT 69 99 94 99

ELISA 57 96 84 99

LeptoTek LFA

66 93 81 96

Zephyr LFA 49 100 83 98


Drawbacks•Confirmation by MAT

•Based on serology: too la te for early diagnosis

•Differences in diagnostic accuracy in di fferent endemic backgrounds: Local evaluation is needed

•Based on crude antigen?


Rapid vs early diagnosis

Rapid diagnosis with po int-of-care test →

Rapid diagnosis is NOT equa l to ear ly diagnosis →

Early diagnosis detects leptospires or leptospira l products at an early acute phase of disease


‘Early’ tests: Detecting leptospires or products

• DFM

• Capture ELISA

• Observation by staining thin sections

• Immunological detection

• PCR detection of DNA


Detect leptospires or their productsDFM•Sensitiv ity of 104 leptospires per ml•Protein fibers mimic leptospires, low specificity: NOT recommended

Antigen capture tests•sensi tivi ty of about 105 leptospires (equivalents) per ml•Improved detection assays not to be expected within years to come

Staining of thin sections• silve r staining• immunohistochemistry

Useful for , e.g., kidney carriers, but limited diagnostic use

Immunological detection• immunofluorescence• radioimmunoassay

Sensitiv ity in the order of 105 per ml


Detect leptospires or their products: PCR

Levels of Specificity•Genus•Species•Serovar

Choice of primers

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Some approaches that have been used

Reistetter, 2006. ompL1 gene – differentiates 6 spec ies

Woo et al., 1997. 23S rRNA gene – differentiates L.biflexa vs L.interrogans

Brown et al., 1997. AP-PCR – roughly at serovar level

Zuerner et al., 1995. IS1533 – Leptospira spec ific but not enough strains tes ted to draw conclusions about spec ies or serovar

Merien et al., 1992 (and many others). 16S rRNA – genus or

spec ies level depending on primers

In real-time PCR: multiplex reactions (Taqman) of differences in Tm (SYBR Green)


Detect leptospires or their productsConv ention al PCR


PCR Phases in Log view


1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

Conventional PCR

No Le ptospira strain 1 H6 10- 3

2 H6 10- 4

3 H6 10- 5

4 H6 10- 6

5 H6 10- 7

6 lely 10- 2 7 1342k 1 0-2

8 1161 1 0-2

9 Cellod 10- 2

10 1051 1 0-3

11 A23 10- 2 12 I CF 10- 3

13 P at oc N.P

14 100bp marker

15 BUT6 N.P 16 H20 N.T

– Agarose gel electrophoresis- Hybridisation(Nested primers)


Primer set SecY II/IV

Primer set Gf3G1a

Probes:

DIGprobe-1DIGprobe-2

G195-28

Conventional PCR: Southern Blotting


Conventional PCRHow does PCR perform?

Brown et al ., 1995. Blood/urine.PCR 62% pos, culture 48% pos.

Mer ien et al ., 1995. PCR vs MAT in 200 cases.14/14 later seroconverted. Another 13 positive?

Yersin et al ., 1999. PCR vs MAT in 90 cases.MAT 75/90 pos, PCR pos 33/75 (44%)

15 MAT neg were PCR pos?

Zhang et al ., 1993. SerumPCR 75% pos but controles 15% pos

There are many more papers in the li terature which describe some use of PCR for diagnosis

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Conventional PCR

• Conventional PCR can be very sensitive

• Contamination leading to false positives is a real problem

• Standard PCR precautions are mandatory

• Separate “clean” rooms for specimen reception etc. etc.


Time consuming

Low diagnostic sensitivity

Size-based discrimination (without hybridization)

Non–Automated

Ethidium bromide

Complicated quantification.

Further limitations of End-Point

(conventional) PCR


Detect leptospires or their productsReal-time PCR



Real-Time PCR Chemistries

Hydrolysis probes

• Molecular beacons/TaqMan Probes

DNA-binding agents

• SYBR green


TaqMan probe

5’ exonuclease activity of the enzyme cleaves the probe

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PCR

Unbound SYBR Green1

Bound SYBR Green1

SYBR Green Dye


AdvantagesLower initial costMelt curve analysisSimple assay design

Disadvantages• Extensive optimisation• Normally used in singleplex reactions

(molecular beacons: high specificity by specific probe and multiplex reactions possible)

SYBR Green PCR


Real-time PCRSmythe et al., 2002. 23S rRNA – Taqman PCR.

Quantitative and improved specificity.Detection threshold 2-10 leptospires (depending on sample type). Sera, Useful

Pa lanaippan et al., 2005. lig-based Taqman PCR.

Detection threshold 6 leptospires.

Mer ien et al., 2005. LAO322-based SYBR Green PCR. Detection threshold 50 leptospires/ml.Sens 49% (25/51 cases).

Levett et al., 2005. lipL32-based SYBR Green PCR.Detection threshold 1-10 leptospires (depending on sample type).

Stoddard et al., 2009. lipL32-based Taqman PCR.

LOD about 10 copies


Real-time PCRReal-time PCR validations; culture as gold standard

•Slack et al., 2007. Taqman rrl- adapted• Analytical sensitivity: 10 copies• 236 samples with 28 culture positives.• Clinical sensitivity 96.4% and specificity 99.5%

•Ahmed et al., 2009. secY – adapted SYBR Green• Analytical sensitivity: 1 – 30 copies, depending on sample type and ‘heterologous’ primer annealing• 114 samples with 12 culture positives• Clinical sensitivity 92% (100% till day 4) and specificity 94%.

•Thaipadunpanit et al., 2011. Compares Taqman rrs with Taqman lipL32-based PCR. Real time PCR contributes to individual patient care


Real-time PCR

• Apart from early diagnosis

• Quick and quantitative

• One tube system; less contamination prone

Useful


Conclusion

• Conventional tests many drawbacks.

•Tests based on serology can be rapid but confirm leptospirosis afterwards . HOWEVER, contribute as such on increasing the awareness.

•Real-time PCR is a promising method for early confirmation of clinical diagnosis when antibiotic treatment is most effective (first 5 days of illness).

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Characterization of Leptospira


Why typing of Leptospira?

• Source identification (same strain in patient and

suspected animal source) → control and intervention

• Immunization; current vaccines (human and animals) give

serovar specific protection – knowledge of local serovars needed for vaccine deve lopment and/or application

• Diagnosis, optimal local MAT panel


Th e epid emiology of leptospirosis is c omplex and dynamic: nearly 300 serovars

Generally adapted to a certain host• More than one serovar adapted to one host

• Pigs: Pomona, Tarrasovi, Bratislava• Cows: Hardjo, Pomona

• One serovar adapted to several hosts• Bratislava: pigs, hedgehogs• Grippotyphosa: common voles, hares

Serovars may adapt to new hosts

Introduction of new animals imply introduction of (new) Leptospira serovars

• Climatologic change• Landuse change (deforestation, urbanization)


Continuous surveillance is important!


Pathogenic and saprophytic leptospires

Saprophytic leptospires live in the environment

Pathogenic leptospires live in the kidneys of host animals


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Survival in the environment

Pathogenic leptospires are excreted with the

urine into the environment

Survival for months if conditions are

favourable•Humid

•Moderate to warm (≤ 30oC)•Neutral to slightly alkaline (pH 6.5 – 8.5)



Picardeau et al., PLoS ONE 2008


Leptospira have to adapt to a wide variety of ‘environments’

• Wide var iety of leptospires; spatial and tempora l

differences

• Wide var iety of clinical manifestations

• Trends of serovars causing mild or severe disease

• Serovars associa ted to cer tain hosts: Identification of

infection source re lies on the determination of the same

leptospire in the patient and in the suspected source


Classification of Leptospira• 2 classification systems

• Based on serology (conventional)• Based on DNA composition/sequence

(recent)

• The two classification systems do not corre late


Leptospira; serological and molecular classification

• Serological classification; serovar is basic taxon• Serovar identified by specific agglutination features with rabbit antisera (CAAT)• >250 pathogenic serovars grouped into 25 serogroups• Typing requires cultured leptospires• Highest titers in MAT give at the best clues on infecting serogroups

• Molecular classification; species• 20 species, including 9 pathogenic• Several methods

• Requiring cultures – e.g. finger printing• Not requi ring cultures – e.g. sequencing of DNA fragments amplified in diagnostic PCR

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Serological characterizationSerogrouping: MAT with rabbit anti-Leptospi ra re ference

seraTyping of serovars: MAT with panels of monoclonal

antibodies

Standard test for serovar character ization is Cross Agglutinin Absorption Test (CAAT); requi res reference

rabbit anti-Leptospi ra antisera and is tedious and labor ious


Molecular characterization


Conventional DNA-based classification m ethods

1. DNA hybridization conditions form the basis for species

determination (Brenner et al., 1999)2. REA, RAPD, LS-PCR

3. PFGE largely coincides with serovar status

Drawbacks• difficult to standardize• No (direct) digital data• Require cultures

International shipment of viable pathogens is di fficult and expensive: Not suitable in most settings


Novel molecular methods

•MLVA (Mul tiple- locus Variable number of tandem repeats (VNTR) Analysis; not genera lly applicable on a ll species (2 of 9

pathogenic spp.)

•(Fluorescent) Ampl ified Fragment Length Polymorphism (F)AFLP; applicable on any organism

•Typing arrays; need for identification of SNPs•(Mul tiLocus) Sequence Typing (MLST); need for conserved

sequences for ampl ification pr ior to sequencing


MLVA

Amplification of short sequence DNA repeats

Number and thus amplicon size strain specific

Need to know the repeats: currently restricted to L. interrogans and L. borgpetersenii

• Majed et a l., J Clin Microbio l. 2005;43:539.• Slack et al., ACMA 2006;4:10

• Na lem et al., PLoS ONE 2010;5:e12637


EcoRIMseI

AdaptersMseI & EcoRI

T4 DN A Liga se

Preselect ive Amplification(p rimers op po site to adapters)

Selective Amplification

(F luo ro ph ore labeled Pr imers withon e base extend ing ou t)

Elec trophoresis

Geno typing

Fluorescent Amplified Fragment Length Polymorphism

Genomic DNA

EcoRIMseI EcoRIMseI

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FAFLP o f Leptosp iral strains

L.inte rrogansL. kirsc hneriL.noguchiiL. weilliL. borgpete rse niiL. santarosai

L. i nte rrogans

L. kirschn e ri

L. santarosai

L. b orgpe te rsen ii

L. n oguch ii

L. we il li

L. i nadai

L. meye ri

L. gen omosp e cie s 1

L. ale xand e riNalam et al., PLoS ONE 2010


Check-Points

multiplex ligase-amplification based typing

array


Pr obes position in the Ar ray Tube

B. Al ig nm en t c on trol .

H. Hyb rid iz ati on c on trol

A. In tern al c on trol

B H 1 2 3 5 6 7 B

8 9 10 11 12 13B 14 15 16 17 20 21 22 25 B

A A A H BB BB H B

B B

Check-Points multiplex ligase probe am plification (MLPA) based typing array

Ahmed et al., Infect Gen Evol, 2010


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MLST of Leptosp iral strains

Ahmed et al., ACMA 2006; Nalam et al., PLoS ONE 2010Thaipa dunpanit et al., PLoS NTD 2007


Summary•Tests based on serology confirm leptospirosis afterwards BUT contribute to increased awareness.

•Culturing leptospires important for epidemiology and for improved MAT panels

•Real-time PCR is promissing for early confirmation of clinical diagnosis when antibiotic treatment is most effective (first 5 days of illness).

•Dawn of isothermal amplification-LAMP?(first report on NASBA in 1994-Colenbrander et al., Ned. Tijdschr Geneeskd).

•CAAT limited to few reference centres

•Conventional molecular typing method not suitable for wide use

•Novel molecular typing methods need only DNA and/or generate digital data for onl ine comparison


Thank you

Documents

Combatting leptospirosis Increased awareness - niah.dld.go.thniah.dld.go.th/th/images/publicnews/033/lepto_file/... · 3 Amsterdam, The Netherlands Some approaches that have been