BRIEF ON LABORATORY

DIAGNOSIS OF VECTOR BORNE

DISEASES

Dr. Md. Ashraf Ali S N

Department of Microbiology

2

MALARIA

RICKETTSIA

DENGUE

CHIKUNGUNYA

Dr Md Ashraf Ali

Laboratory diagnosis of Malaria1. Microscopy

Light microscopy on peripheral blood smear

Fluorescent microscopy

• ACRIDINE ORANGE staining on thin smear

• Quantitative Buffy Coat (QBC)

2. Rapid diagnostic tests (RDT)- Antigen detection

3. Molecular techniques

Dr Md Ashraf Ali

I. MICROSCOPY

1. LIGHT MICROSCOPY

• Thick and thin blood smear- GIEMSA

• Morphological forms of parasite-

demonstrated

2. FLUORESCENT MICROSCOPY

• Stain- ACRIDINE ORANGE (kawamoto

technique)

• Increases the sensitivity

Dr Md Ashraf Ali

Advantage

• Quick, saves time

• Sensitivity more than “PS for MP”.

Disadvantages

• COST: High(capillaries and equipment)

• Species identification and quantification

difficult

Quantitative Buffy Coat (QBC ®)

capillaries

Dr Md Ashraf Ali

II. RAPID DIAGNOSTIC TESTS (RDT)

Antigen detection

Target- pLDH, HRP-2, Aldolase

Formats-

1. Card

2. Dipstick

3. Casette

Low cost- Rs 100-150/- per test

Disadvantage:

low levels of parasitemia cannot be detected

1 2

3

Dr Md Ashraf Ali

III . MOLECULAR TECHNIQUES

1. Polymerase chain reaction (PCR)

Adv: • Can detect low levels of

parasitemia• Can identify species• Drug resistance.Disadv:• Expensive set up• Trained personnel Lane S: Molecular bp standard (50-bp ladder).

Lane 1: P. vivax (size: 120 bp). Lane 2: P. malariae (size: 144 bp). Lane 3: P. falciparum (size: 205 bp). Lane 4: P. ovale (size: 800 bp).

2. Loop mediated isothermal amplification ( LAMP)

Dr Md Ashraf Ali

Summary of malaria diagnostic test

TESTPeripheral

smearQBC

Rapid diag.

testPCR

LAMP

Assay

Sample to be

sent

Finger prick Blood

(EDTA)

Finger prick •finger prick,

•Blood (heparin)

Time taken

(min)30 10 20

Around

5hrs60

Sensitivity 85% 96% 93-97% 76% 98%

SpecificityAbsolutely

specific 93% 95% 100% 99%

Cost

effectiveness• Rs.100 250/- 100-150/- Ref. labs

Dr Md Ashraf Ali

LABORATORY DIAGNOSIS OF DENGUE

GENERAL

• Profound Leucopenia,

• Thrombocytopenia ( < 100,000)

Dr Md Ashraf Ali

MICROBIOLOGICAL1. SEROLOGY- MOST COMMONLY USED

i. Antigen detection

ii. Detection of antibodies

2. MOLECULAR TECHNIQUES- highly specific and sensitive

3. VIRUS ISOLATION- gold standard

Molecular detection and virus isolation done in reference

centres- NIMHANS Bengaluru, NIV Pune.

Dr Md Ashraf Ali

i. ANTIGEN DETECTION

• Sample- serum

• Time- within first 5 days of illness

• Target

o NS1 (non structural protein)

o E/M (structural)

Formats

• Immunochromatographic card test

• NS1 Ag ELISA (Early ELISA)

• NS5 based ELISA also under trialDr Md Ashraf Ali

ii. ANTIBODY DETECTION-

• SAMPLE- Serum

1. ELISA: most widely used

– IgM Antibody Capture (MAC) ELISA

• Detects recent infection

• Sensitivity- 90% and Specificity-98%

– IgG ELISA

Cost- around Rs. 200/- Dr Md Ashraf Ali

Primary V/s Secondary Infection

• Commonly encountered in endemic countries

• Can be differentiated by ratio of specific IgM and IgG

antibody response

• IgM:IgG ratio > 4 indicates Primary infection

• IgM:IgG ratio < 4 Indicates Secondary infection

( re infection)

Dr Md Ashraf Ali

• Other tests- Not routinely used for diagnosis

2. Haemagglutination-Inhibition (HI) assay

3. Neutralization test (NT)

4. Complement Fixation test (CFT)

Dr Md Ashraf Ali

DENGUE COMBO KIT

• NS1 Ag, IgM and IgG detection can be done in a

single kit

Dr Md Ashraf Ali

II. MOLECULAR METHODS

• Detection of viral RNA

• Done on ACUTE PHASE SERA

1. RT-PCR ( RT-Reverse transcriptase)

– real time RT-PCR- 90% sensitive 95% specific

– Nested PCR

2. RT-LAMP

3. NASBA (Nucleic acid sequence based amplification)

Dr Md Ashraf Ali

III. VIRUS ISOLATION

• Samples serum, whole blood(washed buffy coat) Autopsy tissues (liver, spleen)

• Time of collection – within 6days of onset of illness

• Isolation in cell line- mosquito cell line (C6-36)

• Isolation in Suckling mice ( detect antigen in brain by IFA)

after development of encephalitis

• Report- requires a weekDr Md Ashraf Ali

LABORATORY DIAGNOSIS OF CHIKUNGUNYA

Dr Md Ashraf Ali

1. Antibody detection

a) E L I S A- Widely used

b) IgM rapid test

c) Other tests- Haemagglutination Inhibition

2. Detection of viral RNA• RT-PCR

3. Virus isolation

Dr Md Ashraf Ali

ANTIBODY DETECTION

a) MAC ELISA

• Detects Chikungunya specific IgM antibodies

• Sample- serum

• Timing- after 5 days of onset of illness

• Advantage- rapid, accurate.

• Price — range 200/-

Dr Md Ashraf Ali

b) Chikungunya Rapid test

• Sensitivity: 97.1%

• Specificity: 91.1%

• Time: 10 minutes

IgM positiveDr Md Ashraf Ali

LABORATORY DIAGNOSIS OF RICKETTSIA

Dr Md Ashraf Ali

1. SEROLOGICAL TESTS

2. MOLECULAR DETECTION:

– P C R,

– real time PCR

3. ISOLATION OF RICKETTSIAE-

– Cell lines

– Shell vial technique

Reference labsDr Md Ashraf Ali

SEROLOGICAL TESTS

1. Weil- felix

2. Indirect immunofluorescent assay- gold standard

3. ELISA

4. Immunoperoxidase test

5. CFT, Microagglutination test, Haemagglutination

test

Dr Md Ashraf Ali

WEIL FELIX TEST

• Heterophile agglutination test

• Sensitivity- 33%, Specificity- 46%

Advantage

• Inexpensive test

• Acute diagnosis in resource poor settings

Disadvantage

• Not specific

Dr Md Ashraf Ali

E L I S A

• Both specific and sensitive

Disadvantage

• Diagnostic antibody titers are low, during the first

week of illness

Dr Md Ashraf Ali

Facilities available at KIMS

Vector-borne Disease Test

Dengue • NS1 ELISA

• Dengue Combo kit

• MAC ELISA

Chikungunya MAC ELISA

Japanese encephalitis MAC ELISA

Rickettsia Weil-Felix test

Malaria Peripheral blood smear

Dr Md Ashraf Ali

Statistics of vector borne diseases detected in KIMS

January 13 to December 13Name of the test done Total tested Positive

Dengue IgM 1512 555 (36.70%)

Dengue NS1 438 102 (23.83%)

Chikungunya IgM 123 44 (35.78%)

Weil-felix 45 7 (15.56%)

Japanese encephalitis 22 Nil

All the above tests for the diagnosis of vector borne diseases are done free of cost under IDSP (Integrated disease surveillance

project)Dr Md Ashraf Ali

THANK YOU

Dr Md Ashraf Ali