Province Government

Ministry of Social Development

BHAKTAPUR HOSPITAL

Bagmati Province

Bhaktapur

PCR Lab

Operating

Manual

2

Message from the PCR Focal Person

In the field of laboratory medicine, teamwork of paramedical staffs and experts in various

branches of medicine is of paramount importance. Bhaktapur Hospital has undergone

tremendous change in the last six months with advanced technologies and well established

molecular lab is one of them.

I would like to acknowledge Medical Superintendent Dr Sumitra Gautam and Province lab

acting director Mr Narayan Karki also all the dedicated staffs of PCR lab for the devotion and

generous cooperation in development of healthy ambience. With so many overwhelming

responses, what comes to my mind is that “Success comes to those who work hard and devote

their whole life to make a dream come true.”

The input of my staffs has helped to refresh and energize to produce an outstanding result of

molecular lab. The commitment towards the work has brought tremendous enlightenment in both

personal and professional life.

The overwhelming response has been my reward over the years and in my continuous endeavor

to contribute for significant changes and updates. I heartily thank Sujata, Saruna, Renu, Manish

and Rabin for their continuous effort to execute elegant PCR service to Bhaktapur Hospital and

patient.

Sunita Neupane

Focal Person for COVID 19

3

Contents Message from Medical Superintendent ........................................ Error! Bookmark not defined.

List of Acronyms ........................................................................ Error! Bookmark not defined.

1. Introduction .........................................................................................................................4

2. Aims and Objectives ............................................................................................................7

3. Facility design ......................................................................................................................8

4. Equipments, Reagents and Consumables ..............................................................................9

4.1 Equipments ...................................................................................................................... 10

4.2 Reagents .......................................................................................................................... 10

4.3 Consumables ................................................................................................................... 10

4.4 Personnel ......................................................................................................................... 11

5. Staff Details ....................................................................................................................... 12

6. Job Descriptions ................................................................... Error! Bookmark not defined.

7. Workflow ........................................................................................................................... 13

a) Sample Collection .......................................................................................................... 13

b) Sample Processing .......................................................................................................... 14

- RNA Extraction .............................................................................................................. 17

- Master Mix ..................................................................................................................... 21

- Template addition ........................................................................................................... 21

- PCR amplification .......................................................................................................... 24

- PCR analysis .................................................................................................................. 24

c) Reporting ....................................................................................................................... 25

8. Occupational health and Safety Instructions ....................................................................... 31

a) Hospital Waste Management: ......................................................................................... 32

b) Hospital Infection Control: ............................................................................................. 34

4

Introduction

Coronaviruses are a large family of viruses that mostly cause respiratory illness in humans.

Recently identified corona viruses caused illness like Middle East Respiratory Syndrome

(MERS), Severe Acute Respiratory Syndrome (SARS) and, most recently, Coronavirus

Disease19 (COVID - 19). The virus (which was initially called the novel Coronavirus 2019) has

been named SARS- CoV2.

WHO declared COVID-19 outbreak a Public Health Emergency of International Concern on 30th

January 2020 after initial infection seen in Wuhan, China in December 2019. On 11th March,

WHO declared the COVID-19 outbreak as “pandemic”. As of 8th December, 2020, the world has

witnessed 68,117,810 coronavirus cases so far and 1,554,160 deaths. Nepal is also not spared

with the dreary outbreak. The first case in Nepal was reported on 23rd January, 2020. Nepal went

into lockdown for 4 whole months in order to contain the pandemic. But ever since the

withdrawal of the prohibitory orders, the number of new cases in Nepal has been soaring high

with 4-digit number every passing day. (Add latest new case)

1. Test of Corona virus (2019-nCoV)

The gold standard test for SARS-CoV-2 is real-time, reverse transcriptase PCR (rRT-PCR).

Other tests/methods available are antibody test, antigen test, ELISA, CLIA etc.

Polymerase Chain Reaction (PCR) is a laboratory technique used to make multiple copies of a

segment of DNA. PCR is very precise and used to amplify a specific DNA target from a mixture

of DNA molecules. A PCR reaction consists of 3 steps; denaturation, annealing and extension.

5

With a strong history of 115 years, Bhaktapur hospital has been providing access to modern

medicine to the people since decades. At the dawn of pandemic, when only government hospitals

were rendering services to COVID patients, Bhaktapur Hospital was also one of the frontliners.

Bhaktapur Hospital initiated sample collection (Nasopharyngeal / Oropharyngeal) and blood

collection for RDT antibody from Chaitra 10, 2077 (23rd March, 2020). The sample volume

increased drastically so to provide easy access to needy people, it established the molecular lab

for diagnosis of SARS-CoV-2 from Kartik 27, 2077 (12th November 2020). Since its venture into

molecular diagnosis, it has been able to provide service uninterrupted 12 hours a day. To

increase its efficiency and accessibility, sample collection and report dispatch time allocated

from 9:00am to 1:00pm, Sunday to Friday. Validation of COVID-19 RT PCR from NPHL was

obtained on 5th Mangsir, 2077. Besides, proficiency testing samples are obtained from NPHL

and other QC samples from PPHL, Dhulikhel on a monthly basis.

As of 2077/8/26, Bhaktapur Hospital COVID PCR lab has processed 812 samples collected in

the hospital premises itself.

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Table 1: Total tests performed per day

Date Total Samples Positive

2077-07-26 14 5

2077-07-27 27 10

2077-07-28 25 11

2077-07-30 21 8

2077-08-01 9 3

2077-08-02 33 10

2077-08-03 33 10

2077-08-04 36 14

2077-08-05 45 11

2077-08-07 38 11

2077-08-08 43 11

2077-08-09 34 4

2077-08-10 29 8

2077-08-11 28 8

2077-08-12 37 12

2077-08-14 54 14

2077-08-15 37 8

2077-08-16 28 9

2077-08-17 33 7

2077-08-18 25 9

2077-08-19 30 10

2077-08-21 26 4

2077-08-22 26 7

2077-08-23 28 8

2077-08-24 24 4

2077-08-25 21 8

2077-08-26 28 5

Total 812 229

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Figure 1: Total number of PCR tests performed per day

2. Aims and Objectives

The main objective of this manual is to provide guidance to the staffs of COVID-19 lab. It states

the guidelines regarding collection of samples, physical infrastructures, workflow development,

quality assurance and communication of the result.

14

2725

21

9

33 3336

45

38

43

34

29 28

37

54

37

28

33

25

30

26 2628

2421

28

5

10 118

3

10 10

1411 11 11

4

8 8

1214

8 97

9 10

47 8

4

85

2077

-07

-26

2077

-07

-27

2077

-07

-28

2077

-07

-29

2077

-07

-30

2077

-07

-31

2077

-08

-01

2077

-08

-02

2077

-08

-03

2077

-08

-04

2077

-08

-05

2077

-08

-06

2077

-08

-07

2077

-08

-08

2077

-08

-09

2077

-08

-10

2077

-08

-11

2077

-08

-12

2077

-08

-13

2077

-08

-14

2077

-08

-15

2077

-08

-16

2077

-08

-17

2077

-08

-18

2077

-08

-19

2077

-08

-20

2077

-08

-21

2077

-08

-22

2077

-08

-23

2077

-08

-24

2077

-08

-25

2077

-08

-26

Total Samples Positive

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3. Facility design

Laboratory has adequate space and designed in a way to prevent contamination of reactions with

amplified products from previous assays. It has 5 separate rooms allocated:

a) Donning room

b) Extraction room

c) Master Mix preparation and template addition

d) Amplification and product detection

e) Office room

There is a unidirectional workflow to reduce the opportunity of contamination to occur.

- No materials from extraction room should be taken into master mix preparation room.

- No supplies from amplification and product detection room should be taken into

extraction or master mix preparation room.

- A passing door is available in each room to pass the extract or the master mix product.

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4. Progress and planning of Bhaktapur Hospital for testing COVID-19

After establishment of well-equipped molecular lab at Bhaktapur Hospital, it has been rendering

PCR service uninterrupted.

Sample transport

In the initial days when the nation went into lockdown, sample collection facility was made

available in the hospital. There was a dreadful situation when everyone feared of infection.

During those days, samples were sent to NPHL, TUTH, Dhulikhel hospital, Provincial Public

Health Laboratory Dhulikhel and Sukraraj Tropical & Infectious Diseases Hospital. After the

establishment of molecular lab in the hospital’s own premises, all samples are processed here

itself.

Report Dispatch

We use MIDAS Software for report dispatch. Hardcopies are made available to the patients.

However, SMS services and email services are on process for the same.

Laboratory Information System

LIS System currently in use at Bhaktapur Hospital is MIDAS. It gives all the records of test done

in Bhaktapur hospital.

Logistic Management

PCR Machine (BIO-RAD) available: 1

Biosafety Cabinet Level-II (Quest Scientific Technologies): 1

Laminar Hood (Quest Scientific Technologies): 2

Centrifuges (Remi): 2

Serological water bath: 1

Refrigerator: 3

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5. Equipments, Reagents and Consumables

4.1 Equipments

Quality assurance of all the equipments used in PCR is to be done lately. A list of equipments

used is shown below:

1) Thermocycler (BIO-RAD)

2) Centrifuges

3) Biosafety cabinet

4) Laminar flow cabinet

5) Pipette (Accumax)

6) Temperature dependent instruments: Refrigerators

User Log and Temperature log of every instrument is to be maintained daily.

4.2 Reagents

- All reagents are to be maintained contamination free.

- All reagents should be clearly labeled with name, expiration date, relevant safety information.

-Opening date, expiration date and initials of individual should be mentioned on each reagent.

- Reagents from different lot numbers should not be exchanged.

- A copy of manufacturer’s specification and procedures should be kept in SOP binder.

- Logbooks should be maintained for all reagents and kits including product name, manufacturer,

product number, lot number, associated dates of receipt, preparation, open, expiration etc.

4.3 Consumables

Disposable materials used in PCR analysis include pipette tips, eppendorf tubes, PCR tubes and

gloves.

- Special tips should be used for PCR analysis including barrier tips and aerosol resistant

tips.

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- Laboratories should use polypropylene tubes that are lot-certified, RNase-free, DNase-

free and pyrogen-free.

- The size and style of PCR tubes should be as per recommended by manufacturer for

thermocycler.

- Disposable powder free gloves should be available in each section of laboratory.

- Gloves should be interchanged before leaving and entering each section of laboratory.

4.4 Personnel

- Adequate trained staff is required to perform the tests.

- Analysts should have adequate qualifications.

- Personnel responsible for verification must have at least Master’s degree.

-Proper orientation should be given by a trained staff.

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6. Staff Details

S.N. Name Designation

1 Dr. Pramila Vaidhya Consultant Pathologist

2 Sunita Neupane Focal Person

3 Saruna Prajapati Medical Lab Technologist

4 Sujata Baidya Medical Lab Technologist

5 Renu Baga Lab Technician

6 Manish Prajapati Lab Technician

7 Rabin Koju Lab Assistant

8 Dipa Thapa Attendant

Photograph 1: PCR lab team

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7. Workflow

The workflow in a PCR lab is as follows:

a) Sample Collection

b) Sample Processing

- RNA Extraction

- Master mix

- Template addition

- PCR amplification

- PCR analysis

c) Reporting

a) Sample Collection

Respiratory samples are recommended for real time PCR for diagnosis of

COVID-19

Samples should be collected with synthetic swabs with plastic shaft. Do

NOT use cotton swabs with wooden sticks)

Swabs must be collected in leak proof container with VTM1.

Specimen must be correctly labelled and accompanied with correctly filled

sample referral form.

Specimen that can be delivered promptly to the laboratory can be stored and shipped

at 2-8°C for up to 72 hours.

1 When VTM is not available, 0.9% NaCl (2ml) in screw capped vials

can be used. (References: https://jcm.asm.org/content/jcm/58/6/e00590-

20.full.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295134/)

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Note: Consent has been taken from patient before taking the photo for sample

collection.

15

16

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b) Sample Processing

- RNA Extraction

Nucleic acid of virus is enclosed inside protective capsid and envelop so for PCR analysis

nucleic acid is to be extracted from the virus. It can be done by various methods; heat, enzymatic

or microbeads.

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- This is the process where the laboratory personnel are the most vulnerable so it

should be performed in a Class II biosafety cabinet with negative pressure.

- Use 0.5% hypochlorite followed by 70% ethanol to disinfect the work surface. Do not

use bleach for cleaning pipettes.

- Vortexing should be done within the cabinet.

- Extracted RNA should be stored at -20˚C.

Photograph 2: Extraction Room

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20

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- Master Mix

This is the cleanest step in PCR and should be performed in laminar flow hood. The

amplification reagents are prepared just before the template addition.

- Fluorescent probes are sensitive to light so put off the lights while preparing master

mix.

- Gloves should be changed each time upon entering this area.

- Template addition

It is done in a separate cabinet where the RNA extract is added to the master mix reagent.

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Photograph 3: Template Addition

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- PCR amplification

It should be done in a room separate from master mix area.

- PCR analysis

Analysis is done as per the protocol of PCR reagent used.

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c) Reporting

Reporting of the results is based on the protocol of the PCR reagents used. Most of the reagents

detect N gene, E gene, RdRP gene and ORF 1ab. A panel of combination of these genes are

analyzed to report the results.

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Figure 2: Flow Chart for releasing report*

*Reference: Interim Guidelines for SARS-CoV-2 PCR Laboratories in National Public Health

Laboratory Network Nepal

27

28

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Photograph 4: PCR reporting

1) Storage retention and disposal of specimen

Ensuring quality of result: QC results are documented with every run.

a) Internal QC Materials

- PCR Positive Control: to verify that extraction, PCR master mix preparation and

reagent preparation were done correctly.

- Negative Control: to verify that no contaminating nucleic acid has been introduced

into the master mix or into samples during processing.

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- No Template Control: to verify that no contaminating nucleic acid has been

introduced into the master mix.

b) External Quality Assurance

Participation in Validation program and Proficiency Testing program under NEQAS

conducted by National Public Health Laboratory (NPHL).

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Record Keeping

8. Occupational health and Safety Instructions

32

a) Hospital Waste Management:

Every hospital should take appropriate authorization from competent authorities in waste

management. Detailed plans should be made out for waste management detailing like

segregation, collection, transportation, treatment, disposal, safety precautions and training of

staff within the ambit of waste management handling rules.

WHO estimates:

85% of hospital waste is non-hazardous

10% is infectious

5% is non-infectious

The responsibility of segregation should be with the generator of biomedical waste i.e. doctors,

nurses, technicians etc. (Medical and Paramedical personnel).

The segregation should be done as per the categories applicable mentioned in the rules.

33

34

Figure 3: Effectiveness of Disinfectants on various agents

Appropriate Chemical Disinfectant for COVID 19

Chlorine-based product

70-90% alcohol

Hydrogen peroxide ≥0.5%

Things to be considered while selecting decontaminants

concentration

Resistance

compatibility with other materials present

surface being decontaminated

hazards to humans, animals, and the environment associated with the disinfectant.

Ineffective Disinfection can provide a false sense of security and spread

contamination.

35

Preparation of Disinfectants

a. Calculation of liquid sodium hypochlorite concentration:

Formula 1

[% liquid hypochlorite / % desire liquid hypochlorite] -1 = Total part of water for each part of

sodium hypochlorite

E.g. [5% liquid hypochlorite / 0.5 % liquid hypochlorite] -1 = Total 9 part of water for each part

of sodium hypochlorite

Therefore, one must dissolve one part of sodium hypochlorite and 9 part of water in order to

prepare 0.5% sodium hypochlorite out of 5%.

Formula 2

C1*V1 = C2 * V2

Where, C= concentration and V= Volume

If you want to prepare a 0.5% out of 5% chlorine solution

C1 (available) =5% and C2 (desire) =0.5%

V1 is the volume of the chlorine solution to be added to prepare a 0.5% chlorine solution

V2 is the amount of water to be added to prepare a 0.5% chlorine solution.

C1*V1=C2*V2

5% * V1= 0.5% * 1000ml

V1= 0.5%* 1000ml / 5%

V1= 100ml

Therefore, the amount of chlorine solution added to prepare 1000ml of 0.5% chlorine solution

will be 100ml and water is 900ml

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B. Calculation of chlorine solutions from calcium hypochlorite

[% chlorine desired ∕ % chlorine in hypochlorite powder or granules] × 1 000 = grams of calcium

hypochlorite powder for each liter of water.

E.g. [0.5% chlorine desired ∕ 35% in hypochlorite powder] × 1 000 = 0.0143 × 1 000 = 14.3

Therefore, you must dissolve 14.3 grams of calcium hypochlorite powder in each liter of water

used to make a 0.5% chlorine solution.

WEAR PROPER PPE BEFORE

STARTING Disinfection PROCESS!!!

DISINFECTION OF BIOSAFETY CABINET

Wet sponge/ tissue paper with 0.5% hypochlorite and Wipe down all surfaces.

Again wipe it with 70% ethanol.

Run in-built UV Disinfection programme for 15 minutes.

LABORATORY CLOTHING DISINFECTION

Laboratory clothing for reuse (e.g. scrubs) should be washed with hot water/ detergent solution.

– soaked in 0.05% hypochlorite for 30mins (preferably overnight) for Disinfection

OR

Autoclave at 121 ◦C, 15psi for 15 minutes and then washed through laundry

DISINFECTION OF PAPER

Any kinds of documents accompanying samples are sprayed with 70% alcohol.

Ensure key information is captured on laboratory tracking forms/in database prior to

spraying

DISINFECTION OF SURFACES AND FLOORS

Floor surfaces should be cleaned with 0.5-1% hypochlorite solution.

37

If required, cleaning with detergent should precede hypochlorite cleaning. (Lab areas

with high inflow of staff)

Other surfaces and door handles should be cleaned with detergent and 70% ethanol.

38

39

b) Hospital Infection Control:

Each hospital should have the following organization for prevention and control of Hospital

Acquired Infections (HAI):

1) Infection Control Committee:

The committee formulates policies for infection control. Head of hospital should be the

chairman. Members are representative of medical/surgical (HOD), nursing, engineering,

40

administrative, domestic, pharmacy. Committee should meet every three months or

earlier if required.

2) Function:

Policymaking regarding:

a) Provision of adequate buildings, equipments, isolation facilities etc.

b) Ventilation of operation theatre

c) Standardization of procedures for operation theatre, wards, housekeeping, kitchen,

laundry,

d) Preparation of manuals for procedures like preparative skin preparation, IV infusion

and catheterization, lumbar puncture, wound dressing.

e) Formulation of disinfection policy

f) Antibiotic policy for rational use of antibiotic in therapy andprophylaxis

g) Implementation of biomedical waste management rules in hospital

3) Infection Control team:

The team carries out day-to-day measures for control of infection. Infection Control Officer

is usually microbiologist.

Function:

a) Surveillance of infection to give baseline information of the level of endemic infection in

hospital.

b) Investigation of outbreaks of infection including source of infection with epidemiological

typing

c) Controlling the outbreak by rectifying technical lapses if any

d) Monitoring of procedures

e) Monitoring of hospital staff carriers.

f) Training of staffs

g) Ensure implementation of Universal Precautions in hospital

Universal Precautions

41

Basic Principles:

- Consider all patients as potentially infective

- Use appropriate barrier precautions routinely (gloves, masks, aprons and eye protection)

Procedures:

- Hands should be washed before and after all patients or specimen contact.

- Blood of all patients should be handled as potentially infectious.

- Gloves should be worn for potential contact with blood and body fluids.

- Used syringes should be immediately placed in impermeable container.

- Protective eyewear and mask should be worn if splatter with blood or body fluids is

possible (e.g. bronchoscopy, oral surgery)

- Gowns should be worn when splash with blood or body fluid anticipated.

- Masks should be worn when handling patients of TB and respiratory organisms

Protection against blood borne infections (HBV and HIV)

- Mechanical pipetting should be used.

- Spills should be immediately decontaminated.

- Gloves should be worn during handling of blood and blood products.

- All open wounds should be covered with water tight dressing

- Hands should be washed with soap andwater immediately after exposure to the

specimens.

- Work surfaces should be non-penetrative. Use 1% hypochlorite to decontaminate

surfaces.

- Specimens should be decontaminated with 1% hypochlorite before disposal.

Spillage or surface contaminated by blood or blood products:

- Spillage should be covered with absorbent material after which disinfectant (1%

hypochlorite/bleaching powder 14g/dl) should be poured over it and left 20 minutes.

Waste disposal to be dealt separately.

42

- Tissues, organs or limbs removed during surgery should be incinerated or buried deep

with bleaching powder or lime.