Nepal is racing forward to control, eliminate, or eradicate vaccine preventable diseases (VPD), such as poliomyelitis, measles, Neonatal Tetanus and Japanese encephalitis. To achieve these ends, it is necessary to know the burden for each disease in the population and use this information for planning and monitoring of the immunization program. To determine the burden of disease, surveillance data on VPDs is often obtained from patients based on a clinical disease (case) definition. Laboratory confirmation of cases provides more reliable and valid information.



Laboratory Diagnosis depends solely upon the appropriate selection, collection, transportation, and performance of tests under Good Laboratory Practices, including internal and external quality controls at regular intervals. For collection of appropriate samples by medical personnel (e.g. physicians, laboratory technicians, nurses), personnel should be trained and supervised. Even when the attending medical doctor selects and collects the appropriate sample, the result will depend on appropriate storage and transportation of samples to the laboratory. On occasion, samples must be transported over long distances because the required test is not available at peripheral laboratories, further increasing the difficulty of shipping/transporting the samples. Whether transported over short or long distances, some of the laboratory tests will require specimens to be transported at a specific temperature and quality.

Nepal is one of the developing countries with limited diagnostic facilities in government-operated hospitals and privately operated hospitals and polyclinics. National Public Health Laboratory (NPHL) under Department of Health Services, Ministry of Health and Population provides laboratory confirmation of diseases for Public health purposes. WHO-IPD (formerly WHO-PEN) has been working closely with NPHL since 1998 for strengthening laboratory capacities in surveillance of immunization preventable diseases. WHO-IPD with the input of its Microbiologist and NPHL is strengthening the capacity of laboratory for confirmation of Poliomyelitis, Measles, Japanese encephalitis, and meningitis due to Haemophilus influenzae b. NPHL at present is National Measles Laboratory, National Japanese Encephalitis Laboratory and National Haemophilus influenzae surveillance Laboratory.

Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens Infectious substances

An infectious substance is defined as a substance containing a viable microorganism, such as a bacterium, virus, rickettsia, parasite or fungus, that is known or reasonably believed to cause disease in humans or animals*.

Diagnostic specimens

A diagnostic specimen is defined as any human or animal material including, but not limited to, excreta, blood and its components, tissue and tissue fluids collected for the purposes of diagnosis, but excluding live infected animals.
Diagnostic specimens resulting from medical practice and research are considered a negligible threat to the public health.

Packaging, Labeling and Documentation for Transport

The packaging requirements are determined by the UN and are contained in ICAO and IATA regulations in the form of Packaging Instructions (PI) 602 and 650. The requirements are subject to change and upgrade by these organizations. The current packaging requirements are described below. UN-approved packaging systems are available commercially.

Basic triple packaging system:

1. Primary receptacle: A labeled primary watertight, leak-proof receptacle containing the specimen. The receptacle is wrapped in enough absorbent material to absorb all fluid in case of breakage.

2. Secondary receptacle: A second durable, watertight, leak-proof receptacle to enclose and protect the primary receptacle(s). Several wrapped primary receptacles may be placed in one secondary receptacle. Sufficient additional absorbent material must be used to cushion multiple primary receptacles.

3. Outer shipping package: The secondary receptacle is placed in an outer shipping package which protects it and its contents from outside influences such as physical damage and water while in transit.

• Cold Chain:
   – A system of storing and transporting vaccines at recommended temperatures from the point of manufacture to the point of use.
   – Role: Maintain the potency of vaccines

• Reverse Cold Chain:
   – A system of storing and transporting samples at recommended temperatures from the point of collection to the laboratory.
   – Role: Maintain the potency / integrity of the antibodies / antigens present in the samples

Poliomyelitis (AFP):

Case Definition:

An AFP case is any case of acute flaccid paralysis in a person under 15 years of age for any reason other than severe trauma, or paralytic illness in a person of any age in which polio is suspected.
Within 90 days of paralysis onset an AFP case should be classified as “polio” or “polio- compatible”, or "discarded" as non-polio AFP. The WHO case classification diagram gives the criteria by which AFP cases are classified as confirmed paralytic poliomyelitis or discarded as non-polio AFP.

Cases of AFP are classified as polio if:

• Wild poliovirus was isolated from any stool specimen
• Cases of AFP without isolation of wild poliovirus may be classified as “polio compatible” if:

   • Stool specimens were inadequate
   AND
   • Residual weakness was present 60 days after onset of paralysis or 60 day follow up was not done (due to death or absence)
   AND
   “Expert review committee” concludes that these cases could not be discarded as “non-polio” based on available data

Stool Specimen Collection & Transportation for Virus isolation:

When to collect? …… ASAP!!!
STOOL: Recommended in every AFP case.
Highest concentration within 14 days of onset of paralysis
Reduction in time: Almost no virus 2 months after onset
• Intermittent excretion: requires 2 samples, at least 24 hours apart
• Minimum amount: 5 to 8 grams ie. 2 thumb sizes is adequate quantity for necessary lab tests and to keep back-up to repeat tests.

Specimens should be collected before Outbreak response immunization (ORI) is done.
Role of the Laboratory
The laboratory examines stool specimens & determines:
– Whether poliovirus is present or not
– Type of poliovirus: 1, 2, 3
– Origin of the poliovirus : “wild” or “vaccine” (“intra-typic differentiation)

IMPORTANT: the lab results have to be linked with the clinical findings!

Possible Laboratory Results:



Polio Virus & Temperature:

• Thermo labile
• Repeated freezing and thawing will lead to loss of viral titers
In practice, shipping and storage will last: LESS THAN 72 HOURS: USE WET ICE: 2- 80 C

Packing of Specimen: Containers

– Containers: ANY dry, clean, wide mouthed, leak proof transparent, plastic container (30-60ml size) with screw-cap.
– No need of sterile container
The program provides standardized, leak proof containers. These should be used as much as possible, but stock out should not hamper stool collection.

Packing of Specimen: Labels

Container should have a water proof label with: written in English with indelible ink
– Name of patient
– Date of collection
– Stool specimen number
– EPID number

Shipping of specimen: transport containers

• Use vaccine carriers or special (yellow) specimen containers. These carriers should be used and marked for stool shipment only.
Containers are re-usable! Request for standby container from national level.
• Vaccine carrier will keep cool for 36 hours in outside temperature of up to 430C
• Yellow specimen container will keep cool for up to 3 days.
• Choose most direct route. Maximum transit time 72 hrs
• Avoid arrival during week-ends or on holidays
• Always inform destination about arrival by phone or fax.
• Include in message:
   • flight number/ shipping agent (AWB number)
   • date and time of arrival
   • number of boxes
   • number of specimens with EPID numbers

Shipping of specimens: Tips

• Always put yourself in the place of the receiver!
• Use non-erasable ink
• Always check whether:
   – container is not leaking
   – each container is properly labeled
   – documentation is present
   – ice or icepacks are present
   – destination knows about arrival and is ready to pick up the box
   – Request for acknowledgement of receipt or follow-up

Measles:

Virus responsible: Paramyxoviridae- family
                              Morbillivirus- genus

Clinical case definition:
   – fever
   – Maculo-papular erythematous rash, and either cough or coryza and/or conjunctivitis.
   – Koplik spots on the buccal mucosa during the prodromal phase

Laboratory classification*

Laboratory confirmed:

• Detection of measles-specific (IgM) antibodies in serum
• Isolation of the measles virus from urine / nasopharyngeal aspirates.
• Serologic confirmation of measles is defined as the presence of measles-specific IgM or at least a four-fold increase in measles-specific IgG antibody titre.
Epidemiologically confirmed:
A case that meets the clinical case definition and has epidemiological link to a laboratory confirmed case.

Clinically confirmed: A case that meets the clinical case definition and for which no adequate blood specimen was taken.
Discarded: A suspect case that does not meet the clinical or laboratory definition.

* Laboratory classification may also be used for outbreak investigations.

Role & function of the laboratory in measles control

   • Monitoring and verifying virus transmission
   – Confirmation of the outbreaks
   – Confirmation of the cases
   – Identification of the measles virus strains / genetic characterization
• Monitoring susceptibility profile of the population
   – Determination of age distribution of susceptibility to measles in order that the need for immunization campaigns might be assessed.
   – Evaluation of the impact of mass campaigns

Sample Selection & Collection

• Sample selection :
   – Blood / Serum
      • Translucent White/Yellow Serum – Anti-measles-IgM antibody
   – Urine
      • First passed morning Mid stream Urine- 10-50 ml
      • Virus isolation & Viral genome characterization

Sample collection:

What, How much, Who, When =?
• Blood /Serum: 5 ml - Sterile Vial - Doctor / Nurse / Laboratory personnel
• Timing : First / Single Sample - Day 4 and 28 after rash onset
When the measles IgM capture ELISA gives an equivocal result; the clinician needs to make a definitive diagnosis on an individual patient with an initial negative result (WHO).
• Urine: 10-50 ml – By the patient
   • within 3 days after onset of rash
   • First passed morning - Mid stream Urine
   • Sterile Blue capped plastic container (supplied)

Sample Storage & Transportation:

– Arrange for sending samples to laboratory ASSP
– Urine – Do not freeze, Transfer in a cold box to NPHL through WHO-IPD within 24 hrs.
– Blood – Clot (Tilt for 30 minute) – Separate Serum (centrifugation)–
   – Centrifugation – only after complete clot to avoid hemolysis
   – Overnight –refrigerator (2-80C)- Serum separation - decantation
– Serum – Store at –200C for longer storage
   – Domestic refrigerator (2-80C) for max 7 days
– Transfer in cold box to the laboratory ASSP

Testing at Laboratory

– Serum - IgM capture ELISA – NPHL, Teku – Behring Germany Kit
– Urine – Centrifugation – Sediment + VTM (1ml) – NIH Bangkok
– NIH Bangkok – Urine – Virus Isolation – CPE, FAT
        – PCR – N & H genes
        – Phylogenetic analysis

Japanese Encephalitis:

• Japanese encephalitis- a disease caused by a arbovirus of the family flavivirus
• Single-stranded RNA virus
• Similar to West Nile, Murray Valley, and St. Louis encephalitis.
• Affects the membranes around the brain
• Most JE virus infections are mild (fever & headache) or without apparent symptoms
• 1 in 200 infections results in severe disease (high fever, headache, neck stiffness, disorientation, coma, seizures, spastic paralysis and death
• case fatality rate can be as high as 60% among those with disease symptoms;
• 30% of those who survive suffer from lasting damage to the central nervous system.
• Encephalitis occurs mainly in young children because older children and adults have already been infected and are immune.
• Transmitted by a mosquito vector, most commonly species of Culex.
• The major vector Culex tritaeniorhynchus mosquitoes prefer to feed on large domestic animals and birds.
• The pig is found to be the primary amplifying host.
• Birds, such as herons or ducks, have also been implicated in the transmission of JE. Cattle and buffalo, although infected with JE, have low levels of viremia and are believed not to play a role in transmission.
• In urban settings, the primary vectors are mosquitoes of the Culex pipiens complex which breed in contaminated water (e.g., standing puddles, open sewers, fish ponds).

Clinical Description

Infection with Japanese encephalitis (JE) virus may be asymptomatic, or may cause febrile illness, meningitis, myelitis or encephalitis. Encephalitis is the most commonly recognized presentation, and is clinically indistinguishable from other causes of acute encephalitis syndrome (AES).

Recommended case definition

Clinical case definition

Clinically, a case of Acute Encephalitis Syndrome (AES) is defined as a person of any age, in any geographical region, at any time of year with the acute onset of fever and a change in mental status (including symptoms such as confusion, disorientation, coma, or inability to talk ) AND/OR new onset of seizures (excluding simple febrile seizures).

Case classification

Suspected Case: A case that meets the clinical case definition

Laboratory-confirmed JE: A suspected case that has been laboratory-confirmed as JE.

Laboratory criteria for diagnosis

Presumptive:

Detection of acute phase anti-viral antibody response through one of the following:

Elevated and stable serum antibody titters to JE virus through ELISA, haemagglutination-inhibition or virus neutralization assays or IgM antibody to the virus in the serum

Confirmatory:

1. JE virus-specific IgM in the CSF, or Serum*
2. Four fold or greater rise in JE virus-specific antibody in paired sera (acute and convalescent phases collected during first day of admission and at discharge or serum samples collected on 4th day of start of illness and on 10th day of illness, with at least 7 days interval) through IgM / IgG ELISA, haemagglutination inhibition test or virus neutralization test, in a patient with no history of recent yellow fever vaccination and where cross-reactions to other flaviviruses have been excluded, or
3. Detection of JE virus, antigen or genome in tissue, blood or other body fluid by immuno-chemistry or immuno-fluorescence or PCR.
4.
* Detectable level of anti-JE IgM antibody in Serum is confirmatory for surveillance Purpose.

The standard of JE diagnosis in practice is IgM-capture ELISA using CSF or serum samples.

Note: JE infections are common and the majorities of them are asymptomatic. JE infections may occur concurrently with other infections causing symptoms of involvement of central nervous system. So, only serological evidence of recent JE viral infection may not be correct in indicating JE to be the cause of the illness.

In order to confirm a case of JE a patient must have clinical evidence of JE infection in addition to positive serology. Identification of JE IgM in CSF indicates the presence of JE virus in the central nervous system. A definitive diagnosis of JE can be made with viral isolation from cerebral spinal fluid (CSF) or, in fatal cases, CNS tissue; but viral isolation is not possible in most cases due to low titers and rapid neutralizing antibody. Seventy-five percent of CSF or serum samples will be IgM positive at four days after onset of fever, but almost all samples drawn one week after presentation will be positive.

The CSF will usually have a pattern that is consistent with a viral infection; slightly elevated protein, normal glucose, and lymphocyte pleocytosis. CSF is desired for diagnosis as it confirms a CNS infection and has higher titers than serum, however, a single serum specimen drawn at least 7 days after the onset of fever in a patient with a clinical syndrome consistent with encephalitis can be diagnostic. Paired sera tested for total antibody can also be used for diagnosis looking for a 4-fold rise in titer between acute serum, drawn at presentation, and convalescent serum, usually drawn 2 weeks after the acute serum. Difficulty in diagnosis arises in areas where several Flaviviruses coexist. There is significant cross reactivity among Flaviviruses, particularly IgG, so that a Dengue and West Nile infection can give a small increase in JE titers and vice versa. It may be necessary to test for Dengue and West Nile in areas where these viruses co-circulate.

Other diagnostic tests are available but are slowly being replaced by the ELISA. Hemagglutination inhibition tests (HI) are not as sensitive or specific as IgM capture ELISA and have cross reactivity with other Flaviviruses, plaque reduction neutralization tests (PRNT) are time consuming and difficult to perform, and PCR is primarily only in use for research purposes. Other newer diagnostic tests include dot blot assay, dip stick and immunofluorescein slide test.

Selection, Collection and Transport of samples for JE diagnosis:

Selection & Collection:

A) Cerebro-spinal Fluid (CSF)

     • CSF is the most preferred sample for the diagnosis of JE.
     • 2-3 ml of CSF collected in a sterile vial
     • CSF collected by the attending medical doctor at the hospital should be sent to NPHL through SMO network maintaining reverse cold chain.