Malaria Diag Manual

8/10/2019 Malaria Diag Manual

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(Laboratory Manual for Malaria Diagnosis)

2552


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2534 2545

Plasmodiumknowlesi


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.. 2545 (Plasmodium knowlesi)

(checking-rechecking) (profi ciency testing)

2552

I


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II

.. .

.


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I II III IV V

VI1 12 93 134 54 59

III


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1 schizont 6 merozoite2 Methylene blue phosphate 243 4 42

4 43

IV


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1 42 103 144 155 176 18

7 Giemsa 228 239 fi eld stain 2710 ( ) 2711 early trophozoite (ring form) 2912 late trophozoite (Amoeboid form) 29

13 Immature Schizont 3114 Mature Schizont 3215 Gametocyte 3216 Plasmodium falciparum 3517 Plasmodium vivax 3718 Plasmodium malariae 3919 Plasmodium ovale 4120 Plasmodium knowlesi 4921 ring form P. knowlesi Giemsa 5022 late trophozoite 51 P. knowlesi Giemsa23 schizont P. knowlesi Giemsa 5224 gametocyte 53

P. knowlesi GiemsaV


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Sporozoite Merozoite (schizogony ) sporozoite Schizogony Sporogony

gamete (gametgony) Exo-erythrocytic stageTrophozoite chromatin

1. Ring form (early and late rings) 2. Amoeboid form (early and late amoeboid)Schizont

Early schizont immature schizont chromatincytoplasm Late schizont mature schizont chromatin cytoplasm

merozoiteExflagellation

(Flagellum) microgametocyte

Microgametocyte

Macrogametocyte

(Sensitivity)

VI


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(Specifi city)

(False positive)

(False negative)

VII


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1

Hippocratis ( ) ( Plasmodium) 6 .. 1880 Laveran

Plasmodium Class Sporozoa ( 1 )

5 1. Plasmodiumfalciparum

2. Plasmodiumvivax 3. Plasmodiummalariae 4. Plasmodiumovale 5. Plasmodiumknowlesi 4 P.falciparum,P.vivax, P.malariae P.ovale(P.ovale 2 3 ) P.falciparum P.vivax P.falciparum P.vivax 0.5%( 2542 0.40% ) 5 Plasmodiumknowlesi

P. knowlesi2543

1


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PCR PCR primers 4 P. knowlesi Giemsa P. malariae

68 (Primary vector) Anophelesminimus,Anopheles dirus Anopheles maculatus

- -

4 2 1. (Sporogony) 2. (Schizogony) 2


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2.1 (Exo Erythrocytic Tissueschizogony)

2.2 (Erythrocytic schizogony)

1. (Sporogony) (1)

(Microgametocyte) exflagellation 8 cytoplasm ( 20 25 ) Flagellum Giemsa

2 ) Exflagellation Exflagellated microgamete

(Macrogametocyte)

chromatin gamete Zygote gametocyte sporogony Zygote 18 24 Zygote Ookinete 18 24 Ookinete


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Ookinete Oocyst Oocyst 40 80 Oocyst daughternuclei Cytoplasm daughter cell Sporozoite 10 15 Sporozoites mature Oocyst Oocyst

Sporozoite hemocoele hemolymph 2% Sporozoite

1


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proboscis Sporozoite human phase

Sporozoite (gametocyte) Sporozoite P.falciparum , P.vivax P.ovale 10 20 P.malariae

2. (Schizogony) 2 2.1 (Tissue schizogony) ( 1 )

intrinsic phase Sporozoite Sporozoite

Exo erythrocyticschizogony Sporozoite Schizont 45 60 cytoplasm merozoite merozoite 1.0 1.8

schizont merozoite 1


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1 schizont merozoite

6 16 merozoite Merozoite P.falciparum P.malariae (Relapse) (recurrent parasitemia)

recrudescence parasite P.vivax P.ovale Sporozoite (relapse) Hypnozoites

2.2 (Erythrocytic schizogony) 1. Merozoite

1.1 Trophozoite stage 2 1.1.1 Early trophozoite

cytoplasm ring form

( )

Schizont()

merozoite( )

P.falciparum

P.vivax

P.malariae

P.ovale

5 - 76 8

12 169

60454570

~ 40,000~ 10,000~ 2,000~ 15,000


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1.1.2 Growing trophozoite cytoplasm amoeboid form haemoglobin pigment haemozoin ( haematin + protein ) Giemsa cytoplasm pigment 1.2 Schizont stage small nuclei

cytoplasm cytoplasm 1 merozoite P.falciparum 18 32 ( 20 ) P.vivax 12 24 ( 16 ) P.malariae 6 12 ( 8 )

P.ovale 6 12 ( 8 ) ( schizont stage ) merozoite merozoite schizogony erythrocytic stage

P.falciparum 36 48 P.vivax 42 48

P.malariae 72 P.ovale 50 ring form mature schizogony erythrocytic schizogony Plasmodium spp.


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schizont schizont schizont pigment

1. ( Relapse ) P.vivax P.ovale merozoite sporozoite 2 1 merozoite schizogony erythrocytic schizogony

2 merozoite hypnozoites schizogony merozoite erythrocytic schizogony 2. 315 merozoite erythrocyticschizogony

gametocyte (male gametocyte microgametocyte) (female gametocyte macrogametocyte) 8 9

sporozoite microgametocyte macrogametocyte P.falciparum 7 trophozoite P.vivax 5 - 7 trophozoite P.malariae 20 trophozoite P.ovale 5 - 7 trophozoite


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2 2

( 2 ) 6 12 2 (rapid diagnostic test;

RDT) dipstick 10 14 / P.falciparum 8 12

P.vivax 10 15

2


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P.malariae 30 40 P.ovale 10 15

2 3 Periodicity

( < 7 )

P.f mixed P.f P.v, P.o, P.m, or P.kn

mixed P.f P. knowlesi

2


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P.falciparum P.malariae hypnozoite relapse 3. Recrudescence P.falciparum 1 P.malariaerecrudescense 4. Reinfection


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. (4, 5)

- ( AIDS Human Immunodefi ciency Virus

HIV ) - ( Hepatitis B Virus HBV )

1. (Blood pricking) - - (Disposable lancet)

( Single use )

3


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- 1 70% 2

- - 2. ( 6 )

- ( 3 )

3

- -

70%


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4

1. 2. ()

3. blood lancet () 4. 5.


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- (.) -

70% Antiseptic 5% ( Haiter ) ( Clorox )

- - 70% Antiseptic

-

- 70%

(Thin film) (5) 1. Syringe capillary 1 .


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2. (spreader)

3045 ()

(Thin film) (5) 1. Syringe capillary 1 . 2. (spreader)

3045 o ()

(www.radil.missouri.edu/.../MakingBloodSmear.asp 14 2552)

(1)

(2)

(3)

(1) capillary (2) 30-45 (3)

5


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3.

4. ( Fixation ) Methyl alcohol ( Haemoglobin )

(Thick film) (6) -

1 x 1 . 10 20 cells / 1

()

()

()

()

() ()

() ()

6


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- ( Fix ) ( Haemoglobin ) - 1 1 (6 ()) fi x fi x

- 2.5 . X 2 .

- 1 - 2 - 3 Fix Solution Acetic acid 0.5% 1 2

( Thin fi lm ) 1.


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2. 3. 40C 6

4.

(Thick fi lm) (Routine) 1. 2.

Methylene blue phosphate . (hemolysis) . (pre-staining)

. Giemsa, Leishmans, Wright stain, Wright-Giemsa stain


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Giemsa Wrights Wright-Giemsa Cytoplasm Hemoglobin Giemsa Wright 1. Giemsa 1.1 Giemsa stain powdered 0.75 gm

Pure methyl alcohol 65.0 ml Pure glycerine 35.0 ml 3 5 . giemsa alcohol 1 3

Stock Giemsa (Magnetic stirrer) 1.2 Buffer

Disodium monohydrogen phosphate (Na2 HPO4) 6 gm Monopotassium dihydrogen phosphate (KH2PO4) 5 gm

buffer 1,100 ml buffer 1:10 1 buffer pH 7.0 7.2


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1.3 (7 8) Fix absolute methylalcohol fi x - stock Giemsa Buffer 10 % (1:9)

Giemsa - 8 10 - Buffer

-

Giemsa1. Stock giemsa Buffer

1 : 9 2. Methyl alcohol fi x 2 3

7 Giemsa


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3. Methyl alcohol 4. 8 10 5. ( ) 6. slide rack ( WHO , 1961 )

1. 2. clot 3. 4. 5. 6. ( WHO , 1961 )

3 1. 2. 3.

8


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Methylene blue phosphate

1. Methylene blue solution 3

2. Buffer

Haemoglobin 3.

Methylene blue phosphate 2

2 Methylene blue phosphate

Methylene blue phosphate 3 2 magnetic stirrer

(.)

1,500 1,000 500 300

Methylene blue 1.0 0.66 0.33 0.20

Di-sodium phosphate

(Na2PHO4)

3.0 2.00 1.00 0.60

Potassium phosphate (KPHO4) 1.0 0.66 0.33 0.20

. 1,500 1,000 500 300


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2. Wrights 2.1 Stock solution Stain (powdered) .....................0.3 gm Glycerin (C.P.) ........................3.0 ml Methyl alcohol , absolute , acetone free . 97.0 ml. glycerin Methyl alcoholMagnetic stirrer 2-3

2.2 2.2.1 (fi x) Methyl alcohol

2.2.2 1-3 () 2.2.3 Buffer pH 7.2

3-5 2.2.4 buffer 3. Leisshmans stain,Fields stain Field

Field stain 2 stain A stain B Stain A Methylene Blue (medicinal) 0.8 gm Azure I 0.5 gm Disodium hydrogen phosphate (Na2HPO4) 5.0 gm


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Potassium dihydrogen phosphate (KH2PO4) 6.25 gm 500 ml Stain B Eosin 1.0 gm Disodium hydrogen phosphate (Na2HPO4) 5.0 gm Potassium dihydrogen phosphate (KH2PO4) 6.25 gm 500 ml Magnetic stirrer (

) 24 Oxidation (Oxidation scum)

9 1. Solution A 5 2. 5 3. Stain B 3 4. 5


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9 field stain

10 , 11 , 12 , 13, 14 15

www.shoklo-unit.com/.../labman/labman_e.html(25 2552)

10

( )


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11 early trophozoite (ring form)

12 late trophozoite (Amoeboid form)

1. P. falciparum( 16 ) - -


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- Maurers dots comma like bricked red 3 8

P. vivax( 17 ) - - - Schuffners dots P. malariae( 18 ) - -

- Ziemanns dots P. ovale(19) - P.vivax(oval shape) (fi mbriae) - Schuffners dots 2. 4 2

2.1 schizogony 2 2.1.1 Trophozoite merozoite chromatin nucleus 1 1) ring form ( 11 ) - early ring form - late ring form malaria parasites merozoite (

) chromatin nucleus cytoplasm (vacuole) 2) amoeboid form ( 12 ) - early amoebo id pseudopod vacuole


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- late amoeboid pseudopod amoeboid form chromatin cytoplasm chromatin cytoplasm chromatin cytoplasm amoeba

2.1.2 Schizont chromatin 1 - early or immature schizont ( 13 ) chromatin

cytoplasm - late or mature schizont ( 14 ) cytoplasm chromatin chromatin (small daughter cells) merozoites (malaria pigment) cytoplasm

13 Immature Schizont


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14 Mature Schizont

15 Gametocyte

2. (Gametocyte) merozoite female gametocyte macrogametocyte male gametocyte microgametocyte Gametocyte


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a. Immature (young gametocyte) ( 15 ) merozoite asexual form

chromatin nucleus 1 cytoplasmvacuole

b. Mature (full growth gametocyte) ( 15 ) female gametocyte male

gametocyte

Plasmodium falciparum

(16) - Malignant malaria - - ring form gametocyte peripheral blood - ring form chromatin 2 - ring form - ring form (multi-infection)

- late ring form schizont (coma) cerebral malaria - merozoite 1632 pigment merozoite P. f

- Hypnozoite relapse Re crudescence - dots Maurers dots - 36 48 - Gametocyte asexual form 23 34 asexual form


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- Gametocyte crescent gametocyte P. falciparum Thin fi lm

1. ring form 5% 2. 10% ring 1 ring Thick fi lm 1. ring form 2,500 : 100

( 1 : 25 ) 2. Schizont


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16


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Plasmodium vivax (17) - Tertian malaria Benign Tertian malaria - - Peripheral blood ring form amoeboid , schizont gametocyte

- cytoplasm amoeboid form vacuole - P.vhypnozoites

relapse - mature schizont merozoites 12 24 merozoites P.v - dots Schuffners dots - 48

- Gametocyte asexual form gametocyte 12 18 - gametocyte


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17


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Plasmodium malariae ( 18 ) - quatant malaria 72 - - pigments ring form - peripheral blood

- mature schizont merozoites 8 ( 6 12 ) pigment daisy (rosette form) merozoites P.m - dots Ziemanns dots - 72

- gametocyte


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18


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Plasmodium ovalae ( 19 ) - Tertian malaria - - P. ovale - (peripheral blood)

- P. ovale hypnozoites relapse P. vivax mature schizont merozoite 6-12 (8 ) rosette form - dots Schuffners dots

- 48-50 - gametocyte P.v P.m - cytoplasm amoeboid form P. vivax 19


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19


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3 4

P.f P.v P.o P.m

-

-

- *

-

- Schuffners dots*

- maurers dots*

- Ziemanns dots*

- 1 *

- peripheral blood

-

- chromatin 2 *

- *- band form*

- crescent form

- merozoites

-

+

-

-

-

+

-

+

-

-

+

+-

+

16-32

+

-

-

+

+

-

-

+

+

-

-

12-24

+

+

+

+

+

-

-

-

+

+

-

--

-

6-12

-

+

-

-

-

-

+

-

+

+

-

--

-

6-12

*


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4

P. falciparum P. vivax P. malariae P. ovale

1.Form

peripheral

blood

2.

Merozoite

3.

Gemetocyte

4.

Pigment

5.

- Ring form

- Gametocyte

form

- 20 Merozoites

( 16 32 )

- Crescent form

- Pigment

Gemetocyte

-

-

- Maurers dots

- Ring from

- Amoeboid form

- Schzont form

- Gametocyte

form

- 16 Merozoites

( 12 24 )

- Spherical shape

- Pigment

P.falciparum

-

-

-

Schuffners dots.

- Ring form

- Band form

- Rosette form

- Gametocyte form

- 8 Merozoites

( 6 12 )

- Spherical shape

- Pigment

Cytoplasm

-

- Ziemanns dots

- Ring form

- Amoeboid form

- Schizont form

- Gametocyte form

- 8 Merozoites

( 6 12 )

- Spherical shape

- Pigment

P.vivax

P.malariae

-

- Schuffners dot


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(Asexualform) P.falciparum

(Parasite density) 3

1. (Standard number of fi elds) 2. (Standard number ofred white blood cells.) 3. 1 .. 3 ()

1. 1+ 4+ 1+ 1-10 100 2+ 10-100 100 3+ 1-10 1 4+ 10 1 = 1 oil fi eld ( 100x)

2. - /800 /1,000 3. 1 (cubic millimeter;cu.mm.) (micro liter; l)


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- (5,000 10,000 /..) 8,000 /.. X 100 ____________________ = _________________________________ / .. 100

400 100 8000 .. Complete Blood Count (CBC) ..

X 400 _________ = ___________

8,000 100

X = 400 X 8,000100

= 32,000/.. X = /..

500 1. (Thick fi lm) (Positive)


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(Negative) 100 5 6 ( 10-20 1 ) (Thin fi lm) 500 15-20

2. 1 1 (Mixed infection) 3. Ring form P.falciparum Gametocyte P.falciparum Pigment

Crescent form P.falciparum Peripheral blood 4. Ring form,Amoeboid form, Schizont form, Gametocyte form P.vivax P.vivax 5. 4

Mature schizont Merozoite 6-12 Merozoite Pigment (Rosetteform Daisy form) Gametocyte Pigment P.vivax P.malariae P. knowlesi

6. 4 5 Mature schizont Merozoites 6-12 P.malariae Merozoites Rosette form Dots P.ovale 7. ( 1- 3 )


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-

- - (rapid diagnostic test;RDT) 8. 7

Neg (Negative) * P.falciparum Ring form F Crescent form Fg Ring form Crescent form F+g * P.vivax, P.malariae, P.ovale (Species)

V, M Ov (Form) * 1 ( Mixed infection)

(species) Fg+v, F+v, F+m 150-200

Plasmodium knowlesiP. knowlesi

(Long-tailed and Pig-tailed macaque)


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.. 1932 Knowles Das Gupta Julius Wagner-Jauregg

(.. 2552) (thin blood fi lm) P. knowlesi

P. falciparum doublechromatin ( ) amoeboid form latetrophozoite band forms P. malariae (vacuole)

stippling Sinton andMulligans Schffner type schizont Sinton and Mulligans stippling

16 merozoites ( 10 ) schizont (pigment) 2-3 merozoites

(microgamete) (macrogamete)


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20


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P. knowlesi 21-24

ring form P. knowlesi Giemsa

http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Malaria_il.htm

( 24 2552)

21


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late trophozoite P. knowlesi Giemsahttp://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Malaria_il.htm

( 24 2552)

22


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schizont P. knowlesi Giemsahttp://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Malaria_il.htm

( 24 2552)

23


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gametocyte P. knowlesi Giemsahttp://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Malaria_il.htm

( 24 2552)

24


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4.1

1) (Quantity Control ofBlood Film Examination)

100 6

100 . 60 / (7 ) . 30 /

. 30 /

2) (Quality Control ofBlood Film Examination)

4


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(checking) (rechecking) 99

. (checking)(

) 10 (checking) (.) (.)

100 6 . ( rechecking) (checking laboratory) 10 ( )

. - Falsenegative () - False positive ( )

. (Internal Quality Control: IQC) Buffer


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4.2 (proficiency testing; PT) (designated value) 4.2.1 (randomsampling) 10-20

4.2.2 (designated value) 2 1) 3 - 2 3

- 20 (range) 2) (Moleculartechnique) PCR


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4.2.3 4

- = 2

- genus ( species) = 1 - (False negative false positive) = 0

- = 2 - 1 = 1 - 1 = 0

- = 2 - 1 = 1 - 1 = 0

- = 2 - +50 = 1 - +50 = 0


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10 60 . 50

= x 4

= 50 x 4 = 3.33

60

2.5


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1.

2, 2543 1-41.2. 2535- 2541 3. New Concept in Management

of Malaria4. . . 2543

32-36.

5. Gilles HM. Diagnostic methods in malaria. In: Gilles HM and Warrell DA.Bruce- Chwatts Essential Malariology, Third edition, Edward Arnold,

A division of Hodder & Stoughton, 1993, 79-87.6. :

. 2542, 1-47. . 2542, 1-78. Bangkok School of Tropical Medicine. Laboratory Methods. Mahidol

University, 1999., pp 12-13.9. . 2542, 1-5.10. World Health Organization. Manual of the microscopic diagnosis of Malaria. PAN American Health Organization. Scientifi c Publication

No. 46, March 1960, pp 21.11.

59


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Modifi ed Fluorescence Microscopy Interferencefi lter ( MK- P&P method). 2539; 22: 27-36.

12. Tirasophon W, Ponglikitmongkol M, Wilairat P. et al. A novel detectionof a single Plasmodiumfalciparum in infected blood. Biochem

Biophys Res Comm 1991; 175: 175-184.13. CDC, Malaria. Image Library. http://www.dpd.cdc.gov/ dpdx/HTML/ImageLibrary/Malaria_il.htm . ( 24 2552)14. Jongwutiwes S, Putaporntip C, Iwasaki T. et al. Naturally Acquired

Plasmodiumknowlesi Malaria in Human, Thailand. Emerging Infectious

Disease, (journal online). Vol 10 (12), December, 2004; Page 2211-2213.

60


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. ., ., .

( )

- 27

. . . (MT), ., . -

( )- 27


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. ., (MT), .,(Med Sci) . - () -

- ISO/IEC 17025:2005 - 24

.() . () .

- - - ISO/IEC 17025:2005 - - 10


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