(MD MICROBILOGY)

1. POLIO VIRUSES
• PRESENTER:
• DR M NITHYA
• MODERATOR:
• DR. S PAVANI
• ASSOCIATE PROFESSOR
• UPGRADED DEPT OF MICROBIOLOGY, OMC

2. • A 10 year old boy Raju, hailing from labour colony presented to ED with
mild fever, headache, sore throat for previous eight days
accompanied by vomiting for the previous two days
there was brief asymptomatic period of two days
On the morning of presentation, he experienced pain in the right lower limbs,
followed by weakness and inability to walk at the time of presentation without any
sensory symptoms.

4. PATIENT WAS CONSCIOUS, COHERENT AND COOPERATIVE
VITALS – Normal
PULSE RATE 90/min
BP- 130/70
RR 16/min
SATURATION 90% at room air
No paradoxical breathing
No neck flop

5. SYSTEMIC EXAMINATION
CVS , RS , P/A – Normal
CNS – Higher mental functions preserved
Cranial nerves –Normal
MOTOR SYSTEM – Right LL weakness + [1/5]
REFLEXES – Absent
TONE – Decreased
OTHER LIMBS EXAMINATION NORMAL

6. • Patient mother claims that the onset of weakness was sudden, and static
• It has not progressed since last night but she is afraid of that other limbs might
get involved.
• The physician on duty had examined the case thoroughly.
• They called microbiology resident on duty to help in diagnosis.
• Discuss the D/D and probable diagnosis.

7. DIFFERENTIAL DIAGNOSIS
• MOSTCOMMON
 GB SYNDROME
 ACUTE INTERMITTENT PORPHYRIAS
 ACUTE FLACCID PARALYSIS
 SNAKE BITE
 TICK PARALYSIS
• OTHERS
 TRAUMATIC NEURITIS
 TOXIN –DIPHTHERIA AND BOTULISM

8. STOOL SAMPLE
BLOOD SAMPLE
CSF SAMPLE

9. • CSF ANALYSIS FROM BIOCHEMISTRY SHOWED
• Mild leucocytosis (predominantly lymphocytes)
• Slightly raised proteins
• Normal glucose
-- Towards viral infection

10. SAMPLE COLLECTION
 Two samples(8-10 grams or thumb size)24 hr apart
 Within 14 days of onset of paralysis
 Collected in a clean wide mouth bottle –plastic or glass with screw cap
 Sample stored below 8°C
 If paralysis detected after 2 weeks , sample taken upto 60
days from onset
STOOL EXAMINATION

11.  After reaching the laboratory, sample was inoculated on blood agar and
Macconkey agar and incubated @37 degrees and there was no bacterial
growth observed
 Saline and Iodine wet mount was performed and was examined under low
power microscope – no ova and cyst found
 Stool sample was sent in cold chain to one of the national polio network
laboratories for cultivation of virus.
STOOL EXAMINATION

12. INDIA POLIO VIRUS LABORATORY
NETWORK
• For primary isolation of virus, stool sample has to be sent to laboratories under
global polio laboratory networks
• Eight national laboratories constitute the India poliovirus laboratory network.
• All laboratories do primary isolation of poliovirus from stool specimens received
from their defined geographic areas.
• Enterovirus research centre, Mumbai is one of the seven global specialized
laboratories.
• ERC performs intra typic differentiation (ITD) of poliovirus isolates and
sequencing of all wild polioviruses to guide the program.

13. PRIMARY ISOLATION OF POLIOVIRUS IN CELL CULTURE
• About 1gm of stool is homogenized and disinfected with a 10% suspension of
chloroform and phosphate buffer saline.
• The supernatant is collected and inoculated in cell lines.
• Two types of cell lines are used for poliovirus isolation from the stools.
• The RD cell lines which favour growth of all enteroviruses and L20B cell lines
which favour the growth of only polioviruses.
• The use of these two cell lines gives this system a very high sensitivity and
specificity.

14. PRIMARY ISOLATION OF POLIOVIRUS IN CELL CULTURE
• Growth of any virus in these cell lines is revealed by the development of specific
cytopathic effect (CPE) observed under the inverted microscope.
• Normally CPE appears within 7 days of inoculation but if any culture is negative
after 7 days, a blind passage is done on fresh cell lines and watched for another
7 days before declaring the sample negative

15. PRIMARY ISOLATION OF POLIOVIRUS IN CELL CULTURE
• . If the CPE is evident only in the RD cell lines, it is passaged into L20B cell lines
for confirmation of poliovirus.
• If no CPE appears in L20B, the sample is declared as positive for non-polio
enteroviruses (NPEV).
• If CPE appears in L20B cell line the isolate goes for serology (neutralisation test).
• The neutralization test will determine the serotype (type 1, 2, or 3) of the
poliovirus by using poliovirus antiserum.

16. INTRA-TYPIC DIFFERENTIATION TEST (ITD)
• All poliovirus isolates from primary isolation are further tested to determine
whether the particular isolate is wild poliovirus or vaccine poliovirus.
• For ITD two tests are conducted which are ELISA and probe hybridization.
• Both tests must be positive before the result is confirmed.

17.  Threetypesofantibodies
Neutralizing antibodies (IgG)
Antibodies to C antigen (IgM)
Anti-D antibodies
 Complementfixationtest–detects IgM and Anti-D antibodies
Identifies exposure to poliovirus not for type- specific diagnosis
Less often employed
SEROLOGICAL TESTS

18. TREATMENT

19. Symptomaticandsupportive
 Rest in bed
 Relief of pain and spasm of muscles
 Neutral positioning of the limbs
 Physiotherapy
 Good nursing
Treatment

20. Bed Rest
 Essential during acute phase
Physical activity & trauma increases risk of paralytic polio
 Posture to be changed every 2-3 hrs.
 Child to be placed on stomach for short periods each day, to prevent
pneumonia
 Optimum position for limbs
 Hip –slight flexion
 Knee –5 degree flexion
 Foot –90 degree support against the sole
Pain Relief
 Sister Kenny’s treatment
Hot moist packs applied to the muscles to relieve pain and spasm
 analgesics

21. Physiotherapy
Method
 Joints & paralysed muscles – moved passively through full range
 For 10 min , 2-3 times/day
Benefits
 Prevents deformities and contracture
 Promote development of muscle power in non-paralysed muscles

22. Physiotherapy

23. Good Nursing
 Team approach is essential
 Nursing staff is an important part
Diet
 Nutritious , balanced & wholesome
 In non paralytic polio - normal diet
 In paralytic polio
 Fed by Ryles tube
 Calories/kg body wt
 In dysphagia pt. nursed in prone position with foot end raised –gravity
drainage of pooled secretions in pharynx
 Or intermittent suction

24. Rehabilitation
 Physical
 Emotional and Psychological
 Social
 Emotional support to the child helps prepare
himself for better adjustment in life despite the
handicap

26. Immunisation
 History
 Sabin’s Live Polio Vaccine
i. Preparation
ii. Storage and transport
iii. Administration
iv. Dosage
v. Development of Immunity
vi. Advantages and Disadvantages
vii. Complications and Contraindications
 Salk’s Killed Polio Vaccine
i. Preparation
ii. Dosage
 Sabin Vs Salk
 Pulse Polio Immunization

27. History
 By 1953
a. Salk had developed a killed vaccine
b. Almost simultaneously, Koprowsky, Cox and Sabin independently
developed live attenuated vaccines
 Earliest Vaccines
a. Crude Suspensions of spinal cord from infected monkeys
b. Inactivated with Formalin (Brodie and Park)
Ricinoleate (Kolmer)
-Ineffective
-Often dangerous
causing vaccination
poliomyelitis

28. Sabin’s Live Polio Vaccine
 Developed by Albert Sabin
 Sabin’s attenuated strains are developed by plaque selection in MKTC
 Preparation
a. Attenuated strains grown in MKTC
b. Stringent precautions to ensure freedom from SV40 and B virus.
c. Use of molar MgCl2 or sucrose stabilises the vaccine against heat
inactivation

29.  Criteria for selection
a. Should not be neurovirulent
b. Should be able to set up
intestinal infection
c. Should be stable & not acquire
neurovirulence
d. Should possess stable genetic
markers virulent strains
Sabin’s Live Polio Vaccine
As tested by intraspinal inoculation in
monkeys
Following feeding and induce immune
response
After serial enteric passage
Enabling differentiation from wild

30.  Storage:
i. Stabilised Vaccine: 1 Year at 4 °C , 1 month at room temperature
ii. Non-stabilised vaccine: -20 °C in deep freeze (In the freezer
compartment of refrigerator)
 During transport, keep the vaccine under
i. Dry ice (solid carbon dioxide)
ii. Freezing mixture (equal quantities of wet ice and ammonium chloride)
 At vaccination clinic
i. Shouldn’t be frozen andthawed repeatedly  deleterious effect on
potency
ii. Keep vaccine in ice during administration
Sabin’s Live Polio Vaccine

31. Sabin’s Live Polio Vaccine
 OPV in India, trivalent, contains
a. Type 1 – 1lakh TC ID 50
b. Type 2 – 2lakh TC ID 50
c. Type 3 – 3lakh TC ID 50
 Administration – 2drops
 Use the dropper supplied
a. Tilt the child’s back
b. Gently squeeze the cheeks / pinch the nose 
make the mouth open
c. Let the drops fall from the dropper onto the
tongue
Per 0.5 ml
(2drops in India)

32. Sabin’s Live Polio Vaccine
 National Immunization Schedule
AGE DOSE
At birth OPV-0
At 6 weeks OPV-1
At 10 weeks OPV-2
At 14 weeks OPV-3
16-24 months OPV

33. Sabin’s Live Polio Vaccine
 Indian Academy of Paediatrics recommendation
AGE DOSE
At birth OPV-0
At 6 weeks OPV-1+IPV
At 10 weeks OPV-2+IPV
At 14 weeks OPV-3+IPV
16-24 months OPV+IPV
5 years OPV

34. PATHOGENESIS

35. Development of Immunity
Infects intestinal epithelial cells
Replicates  transported to Peyer’s patches
Secondary multiplication subsequent viremia
Spreads to other parts of body
Production of circulating antibodies
Prevents dissemination of virus to nervous system
Prevents paralytic polio
Stimulates production of IgA antibodies
(LOCAL IMMUNITY)
Prevents infection of GIT with wild strains
Vaccine progeny excreted in feces
Non-immunized persons immunized
HERD IMMUNITY
SYSTEMIC IMMUNITY

36. Sabin’s Live Polio Vaccine
 Advantages
i. Oral  easily admin  no need of highly trained personnel
ii. Induces both humoral and systemic immunity
iii. Antibodies quickly produced*
iv. Vaccinees excrete virus  herd immunity
v. Useful in epidemics
vi. Relatively inexpensive
 Disadvantages
i. Instability at high temperatures
ii. Frequent vaccine failures even with fully potent vaccines
iii. Very small residual neurovirulence in OPV

37. Sabin’s Live Polio Vaccine
 Complications
i. Mutation
ii. WHO estimated the risk of
a. Vaccine-associated paralysis : 1 case/million vaccinees
b. Risk of close contact of vaccinee : 1 case/5 million doses of vaccinees
developing paralytic polio
 Contraindications
i. Immunocompromised individuals leukemics, malignacy, those receiving
corticosteroids.
ii. Pregnant mothers  OPV should be delayed until after pregnancy unless
immediate protection is required, when IPV is indicated.
iii. Premature Babies

38. Salk’s Killed Polio Vaccine
 Prepared by Jonas Salk
 Formalin inactivated preparation
 Three types of polio virus grown in monkey kidney tissue culture(MKTC)
Procedure for Preparation
Standard
virulent
strains used
3 types of PVs
grown
separately in
MKTC
Adequate titre
filtered to
remove debris
and clumps
Inactivated
with formalin
at 37°C FOR
12-15 DAYS
Stringent tests
to ensure
complete
inactivation
Three types
are further
pooled
Further tests
for safety and
potency
Issued for
use

39. Salk’s Killed Polio Vaccine
 1954 nationwide field trial (USA): 80 – 90% protection
 1955 – ‘Cutter Incident; over 100 cases of paralytic poliomyelitis occurred in
vaccinees following insufficiently inactivated vaccine

40. Salk’s Killed Polio Vaccine
 Injectable Polio Vaccine (IPV)
a. 1st dose  given at 6 weeks.
b. Immunity sustained by booster doses every 3-5 years thereafter
c. Vaccination of choice among HIV, other immunocompromised states,
pregnant mothers.
 Enhanced potency IPV
a. Produced in human diploid cells
b. Two s/c doses, 4-8 weeks apart, third may be 6-12 months later.
c. Better seroconversion

41. Pulse Polio Immunization - India
Largest public health campaign ever conducted in a single country
 Occurs as two rounds 4-6 weeks apart during low transmission season of polio-
Nov to Feb
 First round- 9th Dec ‘95 and 20th Jan ‘96
 Sudden, simultaneous, mass administration of OPV on a single day
 To all children 0-5 years
 Regardless of previous immunization
 considered as extra doses which act as supplement
 Do not replace the doses during routine immunization
 Children/infants should receive all their scheduled OPV doses.

42. Acute flaccid paralysis surveillance
DEFINITION
Acute flaccid paralysis is defined as sudden onset of weakness and floppiness in any
part of the body in a child < 15 years of age or paralysis in a person of any age in
whom polio is suspected
PURPOSE
o helps to detect reliable areas where poliovirus transmission is occurring.
o helps us to identify areas of priority for focusing immunization activities.
o It is the most reliable tool to measure the quality and impact of polio
immunization activities

43. Acute flaccid paralysis surveillance
 For polio free certification, it is essential to provide evidence to the certification
committee of the absence of wild polio virus transmission through a functioning
and sensitive surveillance system for 3 years after attaining zero polio case status.
Selection of AFP cases for investigation:
 The principle of AFP surveillance is to identify children below 15 years with the
syndrome of Acute Flaccid Paralysis:
Acute: Rapid progression or short, brief duration.
Flaccid: Floppy or soft and yielding to passive stretching at any time
during illness.
Paralysis: Severe loss of motor strength.
Paresis: Slight loss of motor strength.

44. Acute flaccid paralysis surveillance

45. Acute flaccid paralysis surveillance
AFP surveillance after attaining a Zero polio status
 After attaining zero polio cases, it is still critical to continue active
surveillance in all areas in the country to detect any WPV case either
indigenous or importation for at least 3 years after the last confirmed
case.
 This is also a requirement for certification.

46. INDIA AND ITS POLIO STATUS
 India’s last case was reported in West Bengal. The victim,
an 18 month old girl, named Rukhsar from Howrah district
was infected with type 1 poliovirus in January 2011.
 On March 27, 2014, India was declared as polio-free
along with countries of South-East Asian Region of WHO.

50. REFERENCES
 https://www.who.int/features/factfiles/polio/en/
 https://www.who.int/health-topics/poliomyelitis#tab=tab_1
 Reba Kanungo ‘ Ananthanarayan and Paniker’s Textbook of Microbiology
10th edition’ 2017
 Stefan Riedel, Jeffery A. Hobden, Steve Miller, Stephen A. Morse ‘
Jawetz, Melnick, & Adelberg's Medical Microbiology, 28e’.
 Davidson's Principles and Practice of Medicine, International Edition by
Walker, 22nd edition
 file:///C:/Users/nithy/Downloads/Field_guide_for_Surveillance_of_Acut
e_Flaccid_Paralysis_3rd_edition.pdf
 https://www.policeresults.com/national-immunization-vaccination-day-
theme-slogan-quotes-importance-images-observance-awareness-
program/