![DIAGNOSTIC MODALITIES FOR MONKEYPOX WITH ICMR NIV PUNE FOR THE
CONFIRMATION OF MONKEYPOX ON THE SUSPECTED CLINICAL SPECIMENS
a) PCR for Orthopoxvirus genus [Cowpox, Buffalopox, Camelpox, Monkeypox] will be done
b) If specimen will show positivity for the Orthopoxvirus, it would be further confirmed by Monkeypox specific
conventional PCR or real time PCR for Monkeypox DNA
c) Additionally, virus isolation and the Next Generation Sequencing of clinical samples (Miniseq and Nextseq) will
be used for characterization of the positive clinical specimens
All the clinical specimens should be transported to the Apex laboratory of ICMR-NIV Pune routed through the
Integrated Disease Surveillance Programme network of the respective district/state](https://image.slidesharecdn.com/mpxfinal-221216075247-c1495512/85/MPX-final-ppt-25-320.jpg)
MPX final.ppt
- 1. MONKEYPOXVIRUS–AN UPDATE Dr. Paripurna Baruah , MD Consultant Microbiologist Dr. Lal PathLabs, Kolkata
- 2. INTRODUCTION • Zoonotic disease • Genus: Orthopoxvirus • Not similar to Chicken-pox or other pox-like illnesses.
- 4. THE RESURGENCE • Outbreak at a glance • Since 1 January and as of 22 June 2022, 3413 laboratory confirmed cases and one death have been reported to WHO from 50 countries/territories in five WHO Regions. • Description of the outbreak • The majority of laboratory confirmed cases (2933/3413; 86%) were reported from the WHO European Region. • Other regions reporting cases include: the African Region (73/3413, 2%), Region of the Americas (381/3413, 11%), Eastern Mediterranean Region (15/3413, <1%) and Western Pacific Region (11/3413, <1%). • One death was reported in Nigeria in the second quarter of 2022.
- 5. IN INDIA • The 2022 monkeypox outbreak in India is a part of the ongoing outbreak of human monkeypox caused by the West African clade of monkeypox . • 14 July 2022 : Kerala’s State Health Minister announced a suspected imported case which was confirmed hours later by the NIV. • Currently, India has reported 10 confirmed cases of monkeypox and 8 suspected cases: spread across Delhi, Telangana, Bihar and UP. • On 24 July, the first locally transmitted case was reported in Delhi. The individual, a middle-aged male who had no recent history of travelling abroad, was isolated in the Lok Nayak Hospital, New Delhi.
- 8. MPX and SPX • It has been 40 or more years since all countries ceased routine smallpox vaccination with vaccinia-based vaccines. • Whereas smallpox no longer occurs naturally, the global health sector remains vigilant in the event it could reappear through natural mechanisms, laboratory accident or deliberate release. • To ensure global preparedness in the event of reemergence of smallpox, newer vaccines, diagnostics and antiviral agents are being developed. These may also now prove useful for prevention and control of monkeypox. • July 23: WHO declared MPX a PUBLIC HEALTH EMERGENCY OF INTERNATIONAL CONCERN.
- 9. KNOW THE ENEMY • The pathogen • Enveloped double‐stranded DNA virus • genome size :190 kb. • Genus: Orthopoxvirus • also includes vaccinia virus, cowpox virus, variola virus and several other, animal-related poxviruses. • Family: Poxviridae • Two phylogenetically distinct clades of MPXV have been identified through genomic sequencing: • Central African (Congo Basin) clade (CAC) • West African clade (WAC).
- 11. SUSPECTED CASE • A person of any age presenting since 01 January 2022 with an unexplained acute rash or one or more acute skin lesions AND • One or more of the following signs or symptoms • Headache • Acute onset of fever (>38.5 degC), • Lymphadenopathy (swollen lymph nodes) • Myalgia (muscle and body aches) • Back pain • Asthenia (profound weakness) AND • For which the following common causes of acute rash or skin lesions do not fully explain the clinical picture: varicella zoster, herpes zoster, measles, herpes simplex, bacterial skin infections, disseminated gonococcus infection, primary or secondary syphilis, chancroid, lymphogranuloma venereum, granuloma inguinale, molluscum contagiosum, allergic reaction (e.g., to plants); and any other locally relevant common causes of papular or vesicular rash. • Probable case
- 12. PROBABLE CASE • A person meeting the case definition for a suspected case AND • One or more of the following: • has an epidemiological link • direct physical contact with skin or skin lesions of a probable or confirmed case of monkeypox in the 21 days before symptom onset • has had multiple or anonymous sexual partners in the 21 days before symptom onset • has detectable levels of anti-orthopoxvirus (OPXV) IgM antibody (during the period of 4 to 56 days after rash onset); • or a four-fold rise in IgG antibody titer based on acute (up to day 5-7) and convalescent (day 21 onwards) samples; in the absence of a recent smallpox/monkeypox vaccination or other known exposure to OPXV • has a positive test result for orthopoxviral infection (e.g., OPXV-specific PCR without MPXV-specific PCR or sequencing) • Confirmed case •
- 13. CONFIRMED CASE • Laboratory confirmed monkeypox virus by detection of unique sequences of viral DNA by real-time polymerase chain reaction (PCR) and/or sequencing. • Discarded case DISCARDED CASE • A suspected or probable case for which laboratory testing of lesion fluid, skin specimens or crusts by PCR and/or sequencing is negative for MPXV.
- 14. DEFINITION OF A CONTACT • A person who, in the period beginning with the onset of the source case’s first symptoms and ending when all scabs have fallen off, has had one or more of the following exposures with a probable or confirmed case of monkeypox: • direct skin-to-skin physical contact • contact with contaminated materials (fomites) • prolonged face-to-face respiratory exposure in close proximity • respiratory exposure or eye mucosal exposure to lesion material from an infected person • The above also apply for health workers potentially exposed in the absence of proper use of appropriate personal protective equipment (PPE)
- 15. CLINICAL FEATURES PERIOD OF INVASION: • The initial phase of clinical illness typically lasts 1 to 5 days • During which time patients may experience fever, headache, back pain, muscle aches, lack of energy and lymphadenopathy – which is a distinctive feature of this disease INCUBATION PERIOD: 5-21 DAYS
- 16. • PERIOD OF RASH: • Within 1-3 days of appearance of fever. • Face & extremities > trunk • The rash presents in sequential stages • The eruption tends to be centrifugal • Observations from current outbreaks in European and North American countries describe lesions starting in the genital area, but more information is needed
- 17. DIFFERENTIAL DIAGNOSIS • The rash which develops in MPX may resemble other infectious diseases or other conditions, including • Varicella zoster virus (VZV, chickenpox), • Measles, • Herpes simplex virus (HSV), • Primary or secondary syphilis, • Disseminated gonococcal infection (DGI), • Foot and mouth disease, • Chancroid, • Lymphogranuloma venereum (LGV), • Granuloma inguinale, • Molluscum contagiosum, • Scabies, • Rickettsia pox, • Chikungunya, • Zika virus, • Dengue fever, • Vasculitis • other bacterial skin and soft tissue infections.
- 19. 5-21 days Incubation period No symptoms 1-4 days Febrile stage Fever, lymphadenopath y, headache, chills, sore throat, malaise, fatigue 2-4 weeks Rash stage Rash on the skin Days to weeks Recovery
- 20. TRANSMISSION Rspiratory droplets Lesion material Body fluids Contaminated surfaces
- 21. LABORATORY DIAGNOSIS PPE to be donned before collecting the specimens should include- Coveralls/Gowns, N-95 mask, Face shield/safety goggles, double pair of gloves. Donning & doffing of PPE should be carefully performed as per the standard procedure. Testing in a BSLIII and above facility.
- 22. SAMPLE COLLECTION • OPTIMAL SAMPLE: Skin lesions- the roof or fluid from vesicles/ pustules and dry crusts. • Lesion is swabbed vigorously to ensure adequate viral DNA is collected. Both dry swabs and swabs placed in VTM can be used. • 2 lesions of the same type, preferably from different parts of the body should be taken. • All samples being transported should have triple packaging, labelling and documentation.
- 23. • Specimen should be refrigerated or frozen within an hour of collection and transported to laboratory. • If transport/ delay of >7 days, specimen should be stored at <= -20 degree Celsius. • Long term storage (> 60 days) is at -70 degree Celsius.
- 24. SPECIMEN TYPE COLLECTION MATERIAL STORAGE TEMPERATURE PURPOSE Swab of lesion material: • Lesion exudate • Lesion roof • Lesion crust Dacron/ polyester flocked swabs with VTM/ dry swabs Refrigerate (2-8 deg), freeze (-20 deg) within 1 hr of collection, -70 deg or lower after 7 days Recommended for diagnosis Oropharyngeal swab Dacron/ polyester flocked swabs with VTM/ dry swabs See above Recommended for diagnosis if feasible in addition to skin lesion material Rectal/ genital swab Dacron/ polyester flocked swabs with VTM/ dry swabs See above To be considered for research Urine Sterile container See above To be considered for research Semen Sterile container Room temperature (< 1hr), -20 deg after that To be considered for research Whole blood Sterile tube with EDTA Refrigerate (2-8 deg), freeze (-20 deg) within 1 hr of collection, -70 deg or lower after 7 days To be considered for research Serum Serum separating sterile tube See above To be considered for serology to aid diagnosis and research Plasma Sterile tube with EDTA See above To be considered for serology to aid diagnosis and research
- 25. DIAGNOSTIC MODALITIES FOR MONKEYPOX WITH ICMR NIV PUNE FOR THE CONFIRMATION OF MONKEYPOX ON THE SUSPECTED CLINICAL SPECIMENS a) PCR for Orthopoxvirus genus [Cowpox, Buffalopox, Camelpox, Monkeypox] will be done b) If specimen will show positivity for the Orthopoxvirus, it would be further confirmed by Monkeypox specific conventional PCR or real time PCR for Monkeypox DNA c) Additionally, virus isolation and the Next Generation Sequencing of clinical samples (Miniseq and Nextseq) will be used for characterization of the positive clinical specimens All the clinical specimens should be transported to the Apex laboratory of ICMR-NIV Pune routed through the Integrated Disease Surveillance Programme network of the respective district/state
- 26. ICMR- EXPRESSION OF INTEREST • ICMR is willing to make available Monkeypox Virus strain/isolates for undertaking R&D, validation as well as manufacturing activities using characterized isolates of Monkeypox virus under the joint collaboration in the public-private partnership mode for the following two activities– • 2.1 Development of vaccine candidate against Monkeypox disease. • 2.2 Development of diagnostic kits for diagnosis of Monkeypox virus infection https://www.icmr.gov.in/pdf/tender/archive/EoI_version_3.pdf
- 27. • Vero cells. • next generation sequencing. • purified and characterized using the complete genome sequencing. • Bulk preparation of the virus stock • Tissue culture infective dose (TCID50) was estimated. • This virus(inactivated)can be used for the development/validation of diagnostic assays
- 28. MONKEYPOX TESTING IN INDIA • ICMR-NIV, Pune is training 15 VRDLs across the country for testing MPX • AIIMS-Delhi; • Kasturba Hospital for Infectious Disease, Mumbai; • Bangalore Medical College and Research Institute, Bangalore; • Government Medical College in Thiruvananthapuram and • NIV field unit in Kerala; • King Institute of Preventive Medicine and Research, Chennai; and • National Institute of Cholera and Enteric Diseases, Kolkata
- 30. INDIGENOUS KITS IN THE PIPELINE • Genes2me • Trivitron healthcare • Transasia-Erba Biomedicals
- 31. GENES2ME • Specific detection of two distinct Monkeypox virus genetic clades (ie. the central African and westAfrican) along with differentiation from Varicella zoster virus • Primer and Probes are designed using specific and conserved target regions of Monkeypox virus (F3L gene) & VZV (ORF38 gene), enabling specific detection of both Viruses • Skin Lesions – the roof or fluidfrom vesicles, pustules or dry crusts • Compatible with all open Real Time Instruments
- 32. TRIVITRON HEALTHCARE Features • The kit is based upon Single Step RT-PCR, taking place in single tube • Assay developed using four colour fluorescence technology • Different signal signifies the presence of Orthopox group, Monkeypox & Smallpox virus in the sample • Assay validation through the additional human internal control gene • Compatible with both dry swabs as well as sample swabs placed in VTM • A total turnaround time of 1 hour • Compatible with QuantStudio-5/3 and Bio-Rad CFX96, 384 and can also be used on applied Biosystems 7300/7500, Roche Diagnostics LightCycler 96/480, Qiagen Rotor-Gene Q, etc
- 33. • TRANSASIA- ERBA BIOMEDICALS • Highly sensitive but easy to use RT- PCR assay with uniquely formulated primer and probe for enhanced accuracy. https://www.cdc.gov/poxvirus/monkeypox/pdf/pcr-diagnostic-protocol-508.pdf
- 34. ICMR INITIATIVE- VACCINE DEVELOPMENT • New vaccine candidate which is intended to have higher efficacy and safety of the recipients. • ICMR NIV Pune has isolated and characterized MPXV isolate • The genomic analysis was carried out with the whole genomes of MPXV • indicated West African lineage of virus • Genome-wide sequence analysis of the recent outbreak strain of MPXV and other monkeypox viruses • shows a very high conservation • 97.9% (protein-based) and • 97.8% (nucleotide based) sequence identity.
- 35. MEDICAL COUNTERMEASURES CURRENTLY STOCKPILED FOR ORTHOPOXVIRUSES Vaccines • JYNNEOS • ACAM2000 Treatment • Tecovirimat • Vaccinia Immune Globulin Intravenous (VIGIV) • Cidofovir • These countermeasures are not yet widely available. https://www.cdc.gov/mmwr/preview/mmwrhtml/mm5708a6.htm https://www.fda.gov/media/75792/download
- 36. PATIENT MANAGEMENT Principles of Management Patient isolation Protection of compromised skin and mucous membranes Rehydration therapy and Nutritional support Symptom alleviation Monitoring and treatment of complications
- 37. Patient Isolation Isolation of the patient in an isolation room of the hospital/ at home in a separate room with separate ventilation Patient to wear a triple layer mask Skin lesions should be covered to the best extent possible (e.g. long sleeves, long pants) to minimize risk of contact with others Isolation to be continued until all lesions have resolved and scabs have completely fallen off
- 39. MONITORING AND TREATMENT OF COMPLICATIONS • The patient should be closely monitored for the appearance of any of the following symptoms during the period of isolation: • Pain in eye or blurring of vision • Shortness of breath, chest pain, difficulty in breathing • Altered consciousness, seizure • Decrease in urine output • Poor oral intake • Lethargy
- 40. RISK COMMUNICATION AND PREVENTIVE MEASURES Reducing the risk of human-to- human transmission Ambulance transfer
- 41. REFERENCES • https://emergency.cdc.gov/coca/calls/2022/callinfo_062922.asp • https://www.who.int/publications-detail-redirect/WHO-MPX-Clinical-and-IPC-2022.1 • https://main.mohfw.gov.in/sites/default/files/Guidelines%20for%20Management%20of%20Monkeypox %20Disease.pdf • https://www.icmr.gov.in/pdf/tender/archive/EoI_version_3.pdf • https://www.cdc.gov/poxvirus/monkeypox/pdf/pcr-diagnostic-protocol-508.pdf