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Expert Review of Anti-infective Therapy

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Between a bug and a hard place: Trypanosomacruzi genetic diversity and the clinical outcomes of Chagas disease

Louisa A Messenger, Michael A Miles & Caryn Bern

To cite this article: Louisa A Messenger, Michael A Miles & Caryn Bern (2015) Between

a bug and a hard place: Trypanosoma cruzi genetic diversity and the clinical outcomesof Chagas disease, Expert Review of Anti-infective Therapy, 13:8, 995-1029, DOI:

10.1586/14787210.2015.1056158

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Between a bug and a hard

place: Trypanosoma cruzi genetic diversity and theclinical outcomes of ChagasdiseaseExpert Rev. Anti Infect. Ther. 13(8), 995–1029 (2015)

Louisa A Messenger1,Michael A Miles1 andCaryn Bern*2

1Department of Pathogen Molecular

Biology, Faculty of Infectious Tropical

Diseases, London School of Hygiene

and Tropical Medicine, London, UK 2Global Health Sciences, Department of

Epidemiology and Biostatistics, School

of Medicine, University of California

San Francisco, San Francisco, CA, USA

*Author for correspondence:

[email protected]

Over the last 30 years, concomitant with successful transnational disease control programsacross Latin America, Chagas disease has expanded from a neglected, endemic parasiticinfection of the rural poor to an urbanized chronic disease, and now a potentially emergentglobal health problem. Trypanosoma cruzi infection has a highly variable clinical course,ranging from complete absence of symptoms to severe and often fatal cardiovascular and/orgastrointestinal manifestations. To date, few correlates of clinical disease progression havebeen identified. Elucidating a putative role for T. cruzi strain diversity in Chagas diseasepathogenesis is complicated by the scarcity of parasites in clinical specimens and thelimitations of our contemporary genotyping techniques. This article systematically reviews thehistorical literature, given our current understanding of parasite genetic diversity, to evaluatethe evidence for any association between T. cruzi genotype and chronic clinical outcome, riskof congenital transmission or reactivation and orally transmitted outbreaks.

KEYWORDS: cardiomyopathy .

Chagas disease .

congenital transmission .

diagnostics .

genetic diversity. oral outbreaks . reactivation . treatment

Background

Chagas disease is the most important parasiticinfection in Latin America, affecting an esti-mated 5–6 million individuals, with a further70 million at risk [1]. The geographical range of the etiological agent, Trypanosoma cruzi (Kinet-oplastida: Trypanosomatidae), extends fromthe southern USA to Argentinean Patagonia,where it is transmitted by more than 100 species

of hematophagous triatomine bugs (Hemiptera:Reduviidae: Triatominae) [2,3] to at least eightorders of domestic, synanthropic and sylvaticmammalian hosts [4]. Human disease occurswhen infected triatomine feces enter throughintact mucosa or abraded skin [5]. Oral

transmission is an important secondary infec-tion route, responsible for regional microepi-demics of acute Chagas disease in areas oftendevoid of domestic triatomine species, forexample, the Amazon Basin [6]. In recent years,a significant proportion of the infected popula-tion has emigrated from rural areas, leading tothe urbanization of Chagas disease in endemiccountries as well as internationally [7]. Chagasdisease is now considered an emergent global

public health problem associated with congeni-tal transmission [8], blood transfusions [9] andorgan transplantations [10].

Following T. cruzi exposure, human infec-tion begins with an acute phase, lasting up to3 months, during which circulating

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trypomastigotes can be visualized in peripheral blood films orbuffy coat smears. Most individuals are asymptomatic or presentwith a non-specific, self-limiting febrile illness [8]. Mortality dur-ing the acute phase is rare (


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T a b l e 1 . A n o v e r v i e w

o f T r y p a n o s o m a c r u z i h i s t o r i c a l a n d c

o n t e m p o r a r y n o m e n c l a t u r e s .

T e r m

T e c h n i q u e

R e f .

D T U ( c u r r e n t

n o m e n c l a t u r e )

I

I I

I I I

I V

V

V I

[ 3 1 ]

S L - I R ,

2 4 S a

r R N A ,

1 8 S r R N A ,

C y t b ,

H i s t o n

e H 2 B ,

I T S 1 r D N A ,

1 8 S r R N A , g

G A P D H

T c B a t

[ 3 2 – 3 4 ]

P u t a t i v e i n t r a - T c I

s u b d i v i s i o n s

S L - I R

I a ,

I b ,

I c ,

I d ,

I e

[ 4 8 – 5 1 ]

M L M T , m t M

L S T

I D O M

[ 5 2 , 5

3 , 7

0 ]

D T U †

M L E E ,

R A P D

, 2 4 S a

r R N A ,

1 8 S

r R N A

I

I I b

I I c

I I a

I I d

I I e

[ 4 5 , 4

6 ]

Z y m o d e m e

M L E E

Z I

Z I I

Z I I I / Z I A S A T

Z I I I

B o l i v i a n Z I I

P a r a g u a y a n Z I I

[ 1 6 , 3

6 , 3

7 , 1

2 8 , 2

9 0 – 2 9 2 ]

M L E E

Z B

Z A

Z C

Z D

Z B

Z B

[ 2 9 3 , 2

9 4 ]

M L E E

Z 2 ,

5 ,

7 ,

1 0 o r 1 2

Z 4 o r 1 1

Z 1 ,

3 o r 9

[ 2 9 5 ]

I T S - R F L P ,

2 4 S a

r R N A

Z I I I - A

Z I I I - B

[ 2 9 6 ]

B i o d e m e s

M L E E

I I I

I I

I

I

[ 1 7 1 ]

C l o n e t

M L E E

1 – 2 5

3 0 – 3 4

3 5 – 3 7

2 7 – 2 9

3 8 ,

3 9

4 0 – 4 3

[ 3 8 ]

T r y p a n o s o m a c r u z i

T . c r u z i I

T . c r u z i I I

T . c r u z i

T . c r u z i

T . c r u z i

T . c r u z i

[ 4 4 ]

L i n e a g e

2 4 S a

r R N A ,

S L - I R ,

R A P D

2

1

1 / 2

1 / 2

[ 4 1 ]

2 4 S a

r R N A ,

S L - I R

2

1

2 ¢

2 ¢

1

1

[ 4 2 , 4

3 ]

C l a d e

T R ,

D H F R - T S

, C O I I - N D 1

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C

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D

B + C

B + C

[ 6 0 ]

R i b o c l a d e s

2 4 S a

r R N A ,

1 8 S r R N A

2

1

3

4

3

1

[ 2 9 7 ]

H a p l o g r o u p s

M L M T ,

2 4 S a

r R N A ,

C O I I , N D 1 ,

C y t b

Z Z

Y Y

X X

X Y

X Y

[ 5 4 ]

T c M S H 2

A

C

B

[ 2 9 8 ]

G r o u p

K a r y o t y p i n g

C

B

D

A

A

[ 1 5 4 ]

S L - I R

I I

I

I

I

I

[ 2 9 9 ]

R e f e r e n c e s t r a i n

S y l v i o X 1 0 / 1

E s m

c l 3

M 5 6 3 1 c l 5

C a n I I I c l 1

S c 4 3 c l 1

C L B r e n e r

† P r o p o s e d b y [ 4 7 ] .

D H F R - T S : D i h y d r o f o l a t e r e d u c t a s e - t

h y m i d y l a t e s y n t h a s e ; g G A P D H : G l y c e r a l d e h y d e 3 - p

h o s p h a t e d e h y d r o g e n a s e ; M L E E : M u l t i l o c u s e n z y m e e l e c t r o

p h o r e s i s ; M L M T : M u l t i l o c u s m i c r o s a t e l l i t e t y p i n g ; m

t M L S T : M a x i c i r c l e

m u l t i l o c u s s e q u e n c e t y p i n g ; R A P D : R a n d o m

a m p l i f i c a t i o n o f p o l y m o r p h i c D N A ; R F L P : R e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m ; S L - I R : S p l i c e d - l e a d e r i n t e r g e n i c r e g i o n ; T R : T r y p a n o t h i o n e r e d u c t a s e

.

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clades and TcV and TcVI are known to be recent inter-lineagehybrids [30]. As such, TcI–TcIV are characterized by substantialallelic homozygosity, likely resulting from recurrent, dispersed,genome-wide gene conversion, while TcV and TcVI display natural heterozygosity and minimal distinction, sharing intactalleles from their parental progenitors (TcII and TcIII)[54,57,60–62].

Molecular epidemiology of T. cruzi

Molecular epidemiology studies have made substantial progressdefining the phylogeographical and ecological niche of eachT. cruzi lineage (T ABLE 2) [63]. Sylvatic DTU distribution data arestill largely aggregated due to differences in ease of capturebetween reservoir species, paucity of supporting ecologicalinformation and our inability to genotype subpatent zoonoticinfections [64].

In general, TcI, TcII, TcV and TcVI are most frequently isolated from domestic cycles and responsible for the majority

of human infections. TcI has the widest distribution; it is theprincipal cause of Chagas disease in Colombia and Vene-zuela [65–67] and ubiquitous in the sylvatic environment [68,69],primarily circulating in arboreal ecotopes between Didelphis species and the triatomine tribe Rhodniini [70,71], with secondary terrestrial transmission among rodents and sylvatic Triatomaspecies in the inter-Andean valleys of Argentina, Bolivia, Peruand Chile [72–77]. Multiple molecular markers consistently iden-tify high levels of genetic diversity within sylvatic TcI popula-tions [48–51,70,78], and divergent, but genetically homogeneous,strains associated with domestic vectors and humaninfections [52,53,70].

By comparison, TcII, TcV and TcVI are less genetically

diverse overall [30] and appear largely confined to domestictransmission cycles in southern parts of South America [63].The sylvatic reservoirs of these three DTUs are not fully defined, although TcII has been increasingly isolated from pri-mates in Brazil [64,79–81]; peridomestic dogs are emerging aspotential reservoirs of TcV and TcVI in the Gran Chacoregion [82–85]. The geographical range of TcV and VI appearsto be more extensive than previously suggested, with isolatedreports of these hybrid DTUs as far north as Ecuador [86] andColombia [87]. TcIII has a dispersed terrestrial distribution thatextends from northeastern Venezuela to Argentina, where it istransmitted by Panstrongylus geniculatus to Dasypus novemcinctus and other fossorial mammals [88–92]. TcIV is poorly under-stood, principally because several genotyping methods fail todistinguish this lineage from others, especially fromTcIII [42,93,94]. However, TcIV is known to circulate sympatri-cally with TcI in wild primates, Monodelphis and Dasypus spp.in the Amazon [95] and raccoons and dogs in North

America [96]. TcIV is also increasingly detected in human dis-ease, as a secondary agent of Chagas disease in Venezuela [16,66],and in recent oral outbreaks in the Brazilian Amazon [95,93,97–99].

As of now, TcIII and TcIV have only been sporadically detected in domestic transmission cycles, but this may beattributable to undersampling and the limited sensitivity of

some genotyping methods [100]. Finally, TcBat, a new, geneti-cally divergent and potentially human-infective lineage [101],has been isolated from Chiroptera species across Panama [33],Brazil [32] and Colombia [34].

T. cruzi clinical genotyping: perils & pitfalls

Establishing an association between T. cruzi genotype and clini-cal outcome is complicated by inherent biological features relat-ing to parasite infection dynamics, as well as the limitations of our current repertoire of genotyping techniques. Inhumans [53,102–104], triatomine bugs [85,105,106] and mammalianreservoir hosts [88,107,108], mixed infections of distinct parasiteclones are not exceptional but, in many cases, inevitable. Inhighly endemic areas, long-term inhabitants are repeatedly infected by multiple contacts with different triatomines [109],which in turn may have fed on various infected humans and/ormammals, depending on the local disease ecology.

Levels of intra-patient parasite multiclonality might be

expected to increase proportionally with vector exposure. How-ever, this assumes a constant force of infection, incompletecross-genotypic immunity, and lack of genotype interaction(e.g., genotype displacement, reciprocal inhibition, potentiationor recombination) [110–114], transmission population bottlenecks(as observed in related trypanosomes [115]) or any additionalmechanisms that might alter the establishment of secondary infections. The complexity of natural multiclonal parasite pop-ulations is largely unknown and our ability to detect them isrestricted by genetic marker resolution [107,116]. The study of this phenomenon conventionally necessitates deriving biologicalclones from live parasite populations (by micromanipula-tion [117], limiting dilution [118], plating on semi-solid media [106]

or FACS [116]), prior to genetic typing, which introduces a range of potential adaptation biases, discussed below.

Genotyping of T. cruzi can be performed either directly from clinical samples (blood or tissue biopsies) or following parasite isolation by hemoculture or xenodiagnosis. Due to thescarcity of parasites in peripheral blood, especially in chroni-cally infected patients, the former method has limited sensitiv-ity. The primary drawback associated with parasite isolation isselection bias for particular subpopulations, initially by prefer-ential outgrowth due to faster dividing rates and/or culturemedia [55,119,120] and subsequently by loss of clonal diversity from serial maintenance in axenic culture or animals [121–126].Hemoculturing is laborious; recovery rates are usually less than30% among chronic patients [127] and almost entirely deter-mined by parasite load and distribution within the starting sample. Xenodiagnosis, which can facilitate greater parasiterecovery, has also been shown to vary depending on vector per-missibility to local strains [128–130]. Furthermore, due to differ-ential strain tropisms, circulating clones isolated by hemoculture or xenodiagnosis are often genetically distinctfrom those sequestered in tissues [102–104] and can vary evenbetween sequential blood samples [131]. Together, these observa-tions strongly suggest that intra-host parasite diversity is rou-tinely underestimated.

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998 Expert Rev. Anti Infect. Ther. 13(8), (2015)


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T a b l e 2 . A n o v e r v i e w

o f e c

o t o p e s , s y l v a t i c v e c t o r s / h o s t s , g e o g r a p h i c a l d i s t r i b u t i o n s a n d c l i n

i c a l a s s o c i a t i o n s o f t h e m a j o r T r

y p a n o s o m a

c r u z i d i s c r e t e t y p i n g u n i t s .

D T U

E c o l o g i c a l n i c h e

D

o m e s t i c

v e c t o r s

S y l v a t i c

v e c t o r s

S y l v a t i c h o s t s

G e o g r a p h i c a l

d i s t r i b u t i o n

C l i n i c a l f o r m s o f

h u m a n C h a g a s

d i s e a s e

R e f .

T c I

P r i m a r y : l o w l a n d

t r o p i c a l a r b o r e a l

S e c o n d a r y : a r i d

t e r r e s t r i a l

T r i a t o m a d i m i d i a t a

( C

e n t r a l A m e r i c a ) ,

R h o d n i u s ,

P a n s t r o n g y l u s

( B

r a z i l , V e n e z u e l a ,

C

o l o m b i a )

T r i a t o m a ( P e r u ,

B o l i v i a )

P r i m a r y :

R h o d n i u s s p p .

S e c o n d a r y :

T r i a t o m a ,


P r i m

a r y : A r b o r e a l

m a r s u p i a l s ( D i d e l p h i s ) ,

p r i m

a t e s , c a v i o m o r p h s

S e c o n d a r y : T e r r e s t r i a l

r o d e n t s ( P h y l l o t i s

o c i l a e ,

A k o d o n

b o l i v i e n s i s )

P r i m a r y : s o u t h e r n

U S A ,

C e n t r a l a n d

S o u t h A m e r i c a

S e c o n d a r y : c e n t r a l

B r a z i l a n d e a s t e r n

A n d e a n f o o t h i l l s

C a r d i o m y o p a t h y

S p o r a d i c i n

S o u t h e r n C o n e

[ 4 8 – 5 1 , 6

5 , 6

6 , 7

0 , 7

2 – 7 4 , 7

6 , 7

8 , 8

8 , 1

8 5 , 1

8 7 , 3

0 0 , 3

0 1 ]

T c I I

R a r e i n s y l v a t i c c y c l e s

T r i a t o m a i n f e s t a n s


m

e g i s t u s

–

A t l a

n t i c f o r e s t

p r i m

a t e s

A t l a n t i c / c e n t r a l

B r a z i l a n d S o u t h e r n

C o n e


G I m e g a s y n d r o m e s

C o n g e n i t a l

i n f e c t i o n s

[ 6 5 , 7

2 , 7

9 – 8 1 , 1

9 0 , 2

4 3 , 3

0 2 ] ,

T c I I I

T e r r e s t r i a l , f o s s o r i a l ,

l o w l a n d , a r i d a n d

t r o p i c a l

–


g e n i c u l a t u s


l i g n a r i u s

T r i a t o m a

r u b r o v a r i a

A r m

a d i l l o s ( D a s y p u s

n o v e m c i n c t u s ,

E u p h r a c t u s s e x c i n c t u s ,

C h a

e t o p h r a c t u s )

m a r s u p i a l s ( D i d e l p h i s ,

M o n o d e l p h i s ) ,

r o d e n t s , c a r n i v o r e s

N o r t h e a s t e r n

V e n e z u e l a t o

A r g e n t i n a

R a r e i n h u m a n s

[ 8 8 – 9 2 , 3

0 3 ]

T c I V

A r b o r e a l w i t h

t e r r e s t r i a l

t r a n s m i s s i o n i n N o r t h

A m e r i c a

–

R h o d n i u s ,

P a n s t r o n g y l u s ,

T r i a t o m a

P r i m

a t e s , D .

n o v e m c i n c t u s , N a s u a

n a s u a , P r o c y o n l o t o r

S o u t h e r n U S A a n d

n o r t h e r n S o u t h

A m e r i c a

S e c o n d a r y a g e n t i n

V e n e z u e l a

S p o r a d i c o r a l

o u t b r e a k s i n

B r a z i l i a n A m a z o n

[ 1 6 , 6

6 , 6

8 , 9

3 , 9

5 – 9 9 ]

T c V


P u t a t i v e

p e r i d o m e s t i c

t r a n s m i s s i o n a m o n g

d o g s

T .

i n f e s t a n s

–

–

P r i n c i p a l l y S o u t h e r n

C o n e ,

G r a n C h a c o

S p o r a d i c r e p o r t s i n

C o l o m b i a a n d

E c u a d o r



C o n g e n i t a l

i n f e c t i o n s

[ 7 3 , 8

2 – 8

4 , 8

7 , 1

3 5 , 1

6 8 , 2

0 0 , 3

0 4 , 3

0 5 ]

T c V I


P u t a t i v e

p e r i d o m e s t i c

t r a n s m i s s i o n a m o n g

d o g s

T .

i n f e s t a n s

–

–

P r i n c i p a l l y S o u t h e r n

C o n e ,

G r a n C h a c o

S p o r a d i c r e p o r t s i n

C o l o m b i a a n d

E c u a d o r



C o n g e n i t a l

i n f e c t i o n s

[ 8 2 – 8 4 , 8

7 ]

T c B a t

N o t d e s c r i b e d

–

–

C h i r o p t e r a s p p .

P a n a m a , c e n t r a l

a n d s o u t h - e a s t

B r a z i l a n d

C o l o m b i a

O n e i s o l a t e d

h u m a n i n f e c t i o n

[ 3 2 – 3 4 , 1

0 1 ]

D T U : D i s c r e t e t y p i n g u n i t ; G I : G a s t r o i n t e s t i n a l





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T a b l e 3 . O v e r v i e w

o f c u r r e n t a n d h i s t o r i c a l T r y p a n o s o m a c r

u z i g e n o t y p i n g m e t h o d s .

G e n o t y p i n g

m e t h o d

M e t h o d

d e s c r i p t i o n

E x a m p l e o f

g e n e t i c

m a r k e r s

R e p r o d u c i b i l i t y

b / w

a s s a y s

L e

v e l o f

r e

s o l u t i o n

R e a g e n t

c o s t

A d v a n t a g

e s

D i s a d v a n t a g e s

R e f .

M L E E

M e a s u r e s

d i f f e r e n c e s i n

e l e c t r o p h o r e t i c

m o b i l i t i e s o f

i s o e n z y m e s

A S A T ,

A L A T ,

P G M ,

A C O N ,

M P I , A D H ,

M D H ,

M E ,

I C D ,

6 P G D ,

G 6 P D ,

G D ,

P E P ,

G P I

H i g h

D T U l e v e l

I n t r a - l i n e a g e

M o d e r a t e

E a s y v i s u a l

i n t e r p r e t a t i o n

D a t a a m e n

a b l e t o

n u m e r i c a l t a x o n o m i c

a n a l y s i s , f o

r e x a m p l e ,

r a t e s o f s i m

i l a r i t y o r

g e n e t i c d i s t a n c e

R e q u i r e s l a r g e

q u a n t i t i e s o f

p a r a s i t e l y s a t e f r o m

l i v e s t r a i n s

[ 1 6 , 1

2 8 , 1

7 3 , 2

9 3 , 2

9 4 , 3

0 6 ]

R A P D

S h o r t r a n d o m

s e q u e n c e

p r i m e r s u s e d

t o a m p l i f y

u n k n o w n D N A

f r a g m e n t s t o

c r e a t e u n i q u e

b a n d p a t t e r n s

N / A

L o w

D T U l e v e l

L o w

C a n b e p e r f o r m e d

d i r e c t l y o n

f i e l d

s a m p l e s

N o p r i o r s e

q u e n c e

k n o w l e d g e

n e e d e d

U n l i m i t e d n

u m b e r o f

p r i m e r s

D a t a a m e n

a b l e t o


a n a l y s i s


i s s u e s

D o m i n a n t m a r k e r s

m a y c o n c e a l

h e t e r o z y g o s i t y

S t r a i n p r o f i l e s m a y

v a r y w i t h D N A

t e m p l a t e a m o u n t

a n d q u a l i t y

[ 4 0 , 4

1 , 4

5 , 4

6 ]

k D N A - R F L P

R F L P a n a l y s i s

o f k i n e t o p l a s t

m H V R

m H V R

L o w


L o w

H y p e r v a r i a b l e m a r k e r s

C a n p r o d u c e s t r a i n -

s p e c i f i c p r o

f i l e s

R e q u i r e s i s o l a t i o n

o f k D N A f r o m

l i v e

p a r a s i t e s

S t r a i n p r o f i l e

i n h e r i t a n c e m a y

n o t b e s t a b l e o r

c o r r e l a t e w i t h

n u c l e a r t y p i n g

P o t e n t i a l p r o b l e m s

o f c o n t a m i n a t i o n

d u e t o v e r y h i g h

c o p y n u m b e r

[ 1 3 6 ]

k D N A

h y b r i d i z a t i o n

A n a l y s i s o f

m H V R b y

r a d i o a c t i v e

p r o b e


m H V R

L o w

D T U - l e v e l





f i l e s

D N A p r o b e s m a y

c r o s s - r e a c t b / w

D T U s




c o p y n u m b e r

[ 3 0 7 , 3

0 8 ]

a C S D I : A b s o l u t e c h r o m o s o m a l s i z e d i f f e r e n c e i n d e x ; D T U : D i s c r e t e t y p i n g u n i t ; F F L B : F l u o r e s c e n t f r a g m e n t l e n g t h b a r c o d i n g ; G P I : G l u c o s e - 6 - p

h o s p h a t e i s o m e r a s e ; H R M : H i g h - r e s o l u t i o n m e l t i n g ; H S P 6 0 : H e a t s h o c k p r o -

t e i n 6 0 ; k D N A : K i n e t o p l a s t D N A ; L S S P : L o w s t r i n g e n c y s i n g l e s p e c i f i c p r i m e r ; m H V R : M i n i c i r c l e h y p e r v a r i a b l e r e g i o n ; M L E E : M u l t i l o c u s e n z y m e e l e c t r o p h o r e s i s ; M L G : M u l t i l o c u s g e n o t y p e ; M L M T : M

u l t i l o c u s m i c r o s a t e l l i t e

t y p i n g ; m t M L S T : M a x i c i r c l e m u l t i l o c u s s e q u e n c e t y p i n g ; n M L S T : N u c l e a r m u l t i l o c u s s e q u e n c e t y p i n g ; P F G E : P u l s e d - f i e l d g e l e l e c t r o p h o r e s i s ; R A P D : R a n d o m

a m p l i f i c a t i o n o f p o l y m o r p h i c D N A ; R F L P :

R e s t r i c t i o n f r a g m e n t

l e n g t h p o l y m o r p h i s m ; S L - I R : S p l i c e d - l e a d e r

i n t e r g e n i c r e g i o n ; S N P : S i n g l e n u c l e o t i d e p o l y m o r p

h i s m ; S S C P : S i n g l e - s t r a n d e d D N A c o n f o r m a t i o n p o l y m o r p h i s m .

Review Messenger, Miles & Bern

1000 Expert Rev. Anti Infect. Ther. 13(8), (2015)


8/36



u z i g e n o t y p i n g m e t h o d s ( c o n t . ) .

G e n o t y p i n g

m e t h o d

M e t h o d


E x a m p l e o f

g e n e t i c

m a r k e r s


b / w

a s s a y s

L e

v e l o f

r e

s o l u t i o n

R e a g e n t

c o s t

A d v a n t a g

e s


R e f .

K a r y o t y p i n g

( a C S D I )

C o m p a r i s o n o f

c h r o m o s o m e

s i z e v a r i a t i o n

b y P F G E

s e p a r a t i o n a n d

r a d i o a c t i v e

p r o b e


1 F 8 , c r u z i p a n ,

F F A g 6 ,

T c 2 ,

C A 7 . 1

2 ,

C A 7 . 3

2 ,

P 1 9

M o d e r a t e

D T U l e v e l

M o d e r a t e

D a t a a m e n

a b l e t o


a n a l y s i s

R e q u i r e s l i v e s t r a i n s

S t r a i n p r o f i l e s m a y

n o t b e s t a b l e d u e

t o e x p a n s i o n /

c o n t r a c t i o n o f

t a n d e m

r e p e a t s

P r o n e t o

c o n v e r g e n c e b / w

u n r e l a t e d s t r a i n s

[ 1 4 4 , 1

5 4 , 1

5 5 , 3

0 9 ]

D N A

f i n g e r p r i n t i n g

A n a l y s i s o f

v a r i a b i l i t y i n

n u c l e a r

m i n i s a t e l l i t e s

b y r e s t r i c t i o n

d i g e s t i o n a n d

p r o b e


3 3 . 1

5

L o w


L o w




f i l e s



i s s u e s

[ 1 5 6 ]

L S S P - P C R

A n a l y s i s o f

s i z e

p o l y m o r p h i s m s

i n m H V R

a m p l i f i e d b y

L S S P s

m H V R

L o w

D T U l e v e l

L o w

H i g h l y s e n s

i t i v e

C a n b e u s e

d t o d e t e c t

T r y p a n o s o m

a c r u z i i n

i n f e c t e d t i s

s u e s

w i t h o u t p a

r a s i t e

i s o l a t i o n


i s s u e s




c o p y n u m b e r

k D N A s i g n a t u r e s

m a y v a r y w i t h D N A

t e m p l a t e a m o u n t

a n d q u a l i t y

[ 3 1 0 – 3 1 2 ]

S S C P

A n a l y s i s o f

s i z e


i n m u l t i c o p y

g e n e

f r a g m e n t s

S L - I R ,

2 4 S a

r R N A ,

1 8 S

r R N A ,

c r u z i p a i n ,

P 7 - P 8

M o d e r a t e

D T U l e v e l

L o w

R e q u i r e s l i m

i t e d

t e c h n i c a l e x p e r t i s e


D T U a s s i g n m e n t

b a s e d o n p r e s e n c e /

a b s e n c e o f

a m p l i c o n s ;

i n s e n s i t i v e t o

p o t e n t i a l m u t a t i o n s

i n n o v e l s t r a i n s

U n k n o w n i n t r a -

s t r a i n c o p y

h o m o l o g y

[ 2 5 , 3

1 3 , 3

1 4 ]

a C S D I : A b s o l u t e c h r o m o s o m a l s i z e d i f f e r e n c e i n d e x ; D T U : D i s c r e t e t y p i n g u n i t ; F F L B : F l u o r e s c e n t f r a g m e n t l e n g t h b a r c o d i n g ; G P I : G l u c o s e - 6 - p

h o s p h a t e i s o m e r a s e ; H R M : H i g h - r e s o l u t i o n m e l t i n g ; H S P 6 0 : H e a t s h o c k p r o -

t e i n 6 0 ; k D N A : K i n e t o p l a s t D N A ; L S S P : L o w s t r i n g e n c y s i n g l e s p e c i f i c p r i m e r ; m H V R : M i n i c i r c l e h y p e r v a r i a b l e r e g i o n ; M L E E : M u l t i l o c u s e n z y m e e l e c t r o p h o r e s i s ; M L G : M u l t i l o c u s g e n o t y p e ; M L M T : M

u l t i l o c u s m i c r o s a t e l l i t e

t y p i n g ; m t M L S T : M a x i c i r c l e m u l t i l o c u s s e q u e n c e t y p i n g ; n M L S T : N u c l e a r m u l t i l o c u s s e q u e n c e t y p i n g ; P F G E : P u l s e d - f i e l d g e l e l e c t r o p h o r e s i s ; R A P D : R a n d o m

a m p l i f i c a t i o n o f p o l y m o r p h i c D N A ; R F L P :

R e s t r i c t i o n f r a g m e n t

l e n g t h p o l y m o r p h i s m ; S L - I R : S p l i c e d - l e a d e r

i n t e r g e n i c r e g i o n ; S N P : S i n g l e n u c l e o t i d e p o l y m o r p

h i s m ; S S C P : S i n g l e - s t r a n d e d D N A c o n f o r m a t i o n p o l y m o r p h i s m .





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u z i g e n o t y p i n g m e t h o d s ( c o n t . ) .

G e n o t y p i n g

m e t h o d

M e t h o d


E x a m p l e o f

g e n e t i c

m a r k e r s


b / w

a s s a y s

L e

v e l o f

r e

s o l u t i o n

R e a g e n t

c o s t

A d v a n t a g

e s


R e f .

P C R p r o d u c t

s i z e

p o l y m o r p h i s m

A n a l y s i s o f

s i z e


i n m u l t i c o p y

g e n e

f r a g m e n t s

S L - I R ,

2 4 S a

r R N A ,

1 8 S

r R N A ,

A 1 0

H i g h

D T U l e v e l

L o w


d i r e c t l y o n

f i e l d

s a m p l e s


i t e d




a b s e n c e o f

a m p l i c o n s ;

i n s e n s i t i v e t o

p o t e n t i a l m u t a t i o n s

i n n o v e l s t r a i n s

U n k n o w n i n t r a -

s t r a i n c o p y

h o m o l o g y

[ 4 1 –

4 3 ]

P C R - R F L P

R F L P a n a l y s i s

o f m u l t i c o p y

g e n e

f r a g m e n t s

H S P 6 0 ,

G P I ,

C O I I , G P 7 2 ,

1 F 8 ,

h i s t o n e

H 3 ,

I T S ,

T c S C 5 D

H i g h

D T U l e v e l

M o d e r a t e


d i r e c t l y o n

f i e l d

s a m p l e s


i t e d




a b s e n c e o f S N P s ;

i n s e n s i

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