Pemeriksaan Laboratorium nCoV-2019 (COVID-19)

Pemeriksaan Laboratorium nCoV-2019 (COVID-19)

Basti AndriyokoLaboratorium Patologi Klinik RSUP Dr. Hasan Sadikin Bandung

Image from:WHO web

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Specimen Type

Biosafety Level

Safety Procedures During Sample Collection and Transport Lab

Guidance Testing of 2019-nCoV in Reference Laboratories

Diagnostic Knowledge Gaps

Reporting of Cases and Test Results 5

WHO.Laboratory testing for 2019 Novel Coronavirus in suspected human cases.2020

BSL

Biosafety Level 2019-nCoV

• Samples prepared for molecular testing could be handled as would samples of suspected human influenza BSL 2

• Virus isolation in cell culture not recommended, except at a BSL 3 facility

Spec

imen

Typ

e

Specimen TypeSpesimen Jumlah Container

Saluran nafas bagian atas

- Apus nasofaring- Apus orofaring

1 apus1 apus

VTM

Saluran nafas bagian bawah

- Sputum- Tracheal

aspirate- BAL

2-3 ml2-3 ml

Pot sputumSputum trap

Darah - SerumSentrifuse 1000–1300 g 10 minutes

Whole blood 5 ml

Tabung eppendorf

CDC, WHO 2020

Viral Transport Medium (VTM)

Eppendorf tube

www.nursingtimes.net

Pengambilan Spesimen

Apus nasofaring

Apus orofaring

Safe

ty p

roce

dure

s

Safety procedures during sample collection

Ensure that HCWs performing aerosol-generating procedures• Respirators (NIOSH-certified N95, EU FFP2 or equivalent, or

higher level of protection)

• Eye protection (i.e. goggles or a face shield)

• Clean, long-sleeved gown and gloves.


Safe

ty p

roce

dure

s

Safety procedures during sample transport

• Triple layer packaging

Primary tube

Absorbent & Secondary container

Outer packaging

Form Pengantar Spesimen ke Balitbangkes

Test

ing

2019

-nCo

V

• Patients that meet the case definition for suspected 2019- nCoV should be screened for the virus with PCR.

• Laboratories may desire to use a pan-coronavirus assay for amplification followed by sequencing of amplicons from non-conserved regions for characterization and confirmation.

• Several molecular assay have been and are currently under development. Some groups shared their molecular assays, most procedures assume a basic familiarity with PCR/RT-PCR assay.

Testing of 2019-nCoV in reference laboratories


Test

ing

2019

-nCo

V

https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance

In-house developed molecular assays

Test

ing

2019

-nCo

V


In-house developed molecular assays

• China CDC Primers and probes for detection 2019-nCoV (24 January 2020)• Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR –

Charité, Berlin Germany (17 January 2020)• Detection of 2019 novel coronavirus (2019-nCoV) in suspected human

cases by RT-PCR – Hong Kong University (23 January 2020)• PCR and sequencing protocol for 2019-nCoV - Department of Medical

Sciences, Ministry of Public Health, Thailand (Updated 28 January 2020)• PCR and sequencing protocols for 2019-nCoV- National Institute of

Infectious Diseases Japan (24 January 2020)• US CDC panel primer and probes– U.S. CDC, USAV – U.S. CDC, USA (28

January 2020)

Test

ing

2019

-nCo

V


Germany

Corman Victor M,et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3)

• Real-time RT PCR (rRT-PCR) assays for the in vitro qualitative detection of 2019-Novel Coronavirus (2019-nCoV) in respiratory specimens and sera.

• The 2019-nCoV primer and probe sets are designed for the universal detection of SARS-like coronaviruses (N3 assay) and for specific detection of 2019-nCoV (N1 and N2 assays).

CDC.2019-nCoV rRT-PCR Panel Instructions for Use. 2020

Ø Quantitative realtime reverse-transcription PCR (rRT-PCR) assays that can react with multiple coronaviruses that are in the subgenus Sarbecovirus, because there is insufficient public information about the genetic diversity of 2019-nCoV in humans and animals.

Ø Individuals with samples that are positive in these RT-PCR assays should be considered to be infected by the 2019-nCoV

Ø In the event of positive PCR results, sequence analyses to confirm the result and to distinguish between 2019-nCoV and other genetically related coronaviruses.

Ø N gene is recommended as a screening assay.Ø Orf1b assay is recommended as a confirmatory.

Target: ORF1b and N viral genome

Test

ing

2019

-nCo

V

• Menggunakan 2 protokol dari:US CDC : metode rRT-PCR

- Target gen N (Nucleocapsid)

Jerman : metode konvensional- Target gen N (Nucleocapsid) - Target gen RdRp (RNA-dependent RNA polymerase)

• TAT: + 2 hari

Pemeriksaan nCov-2019 di Balitbangkes

Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl G, Wallrauch C, et al. Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany. New England Journal of Medicine. 2020.

Holshue ML, DeBolt C, Lindquist S, Lofy KH, Wiesman J, Bruce H, et al. First Case of 2019 Novel Coronavirus in the United States. New England Journal of Medicine. 2020.

Bastola A, Sah R, Rodriguez-Morales AJ, Lal BK, Jha R, Ojha HC, et al. The first 2019 novel coronavirus case in Nepal. The Lancet Infectious Diseases.

The first 2019 novel coronavirus case in Nepal

Day of illness

2810 26

• Admission to hospital

• Positive rRT-PCR for 2019 n-CoV (throat swabs)

1

Wuhan

6

Nepal

14

Discharged from hospital

Negative rRT-PCR for 2019 n-CoV (throat swabs)

When exactly PCR become negative?

Repo

rtin

g

• Laboratories should follow national reporting requirements.• In line with the International Health Regulations (IHR) (2005),

the national health authority must notify WHO within 24 hours of all events that may constitute a public health emergency of international concern according to defined criteria.

Reporting of cases and test results


Diagnostic Knowledge Gaps

Dia

gnos

tic

Know

ledg

e G

aps

Molecular testing

1. What are the best clinical specimens for optimum RT-PCR yield and identification of cases?

2. What is the excretion pattern of the virus and when is the best time for sampling?

3. How long after recovery do individuals shed the virus, and from which anatomical sites?

4. What is the range of genetic diversity among 2019-nCoV isolates? Will this diversity affect test performance (nucleotide changes in the primer/probe binding region)?

5. Virus adaptation after serial passages in cell culture?https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance

Dia

gnos

tic

Know

ledg

e G

aps

Serological testing

1. What are the best clinical specimens for optimum serological testing of cases/contacts?

2. What is the antibody kinetics (IgM/IgG and neutralizing antibodies) and when is the best time for sampling?

3. What serological tests are available? What are their sensitivities and specificities?

4. Do asymptomatic or mild infections generate antibody response (or the same level as severe infections)?

5. What do negative serological tests mean in asymptomatic/mild/severe cases/contacts (single sample or paired in acute and convalescent) in the absence of validated serological assay?


Kesimpulan:

Dari awal Indonesia bisa melakukan pemeriksaan laboratorium untuk mendeteksi n-CoV 2019

TERIMA KASIH

Pemeriksaan Laboratorium nCoV-2019 (COVID-19)

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