Stool specimens should be processed as soon as possible after arrival in the laboratory. It is assumed that chloroform extraction will have been performed on all specimens within one working day of arrival in the laboratory, and that stool extracts will be available for inoculation onto cell culture. It is not possible however to have cell monolayers ready for inoculation at all times. Therefore, for laboratory management purposes and eventually for certification documentation, it is important to record the date when the first cell monolayers are inoculated. It is also important to record when CPE is first observed in a culture. Standard WHO recommendations require two consecutive passes totalling 14 days of incubation in each cell line used. Isolates obtained from specimens that are negative in L20B cells but positive in RD cells should be repassaged in L20B cells to exclude the possibility that they are polioviruses. Information relating to each of these passes should be recorded separately.
In the past this information was recorded only in laboratory workbooks, but summaries may be required to document laboratory activities for certification. It is now recommended to record this information in a standard database, rather than trying to collect and analyse the information retrospectively. Experience has shown that the easiest way to record information on isolation results is to establish a specimen-based database, with each line of information relating to one specimen. Thus, for a case with two specimens collected and processed there will be two lines of information.
The recommended minimum information to be collected and recorded on specimen processing and isolation results should include the following:
· whether the specimen was processed (y/n);
· date of specimen extraction;
· temperature at which extract stored;
· date of first inoculation onto L20B cells;
· result of first inoculation into L20B cells;
· date of first inoculation onto RD cells;
· result of first inoculation into RD cells;
· date of second inoculation onto L20B cells;
· result of second inoculation into L20B cells and date of result;
· date of second inoculation onto RD cells;
· result of second inoculation into RD cells and date of result;
· date of inoculation of L20B-negative, RD-positive isolate into L20B;
· result for L20B passage and date of result;
· date final isolation result available.
For standard purposes the date of harvesting of cultures and the date of neutralization assays would be recorded only in laboratory workbooks. However, it is possible that in some instances National Certification Committees will require information of this type from the laboratories. Again, it is a good idea to record this information in a standard database immediately rather than trying to collect and analyse the information retrospectively. Experience gained over the years has shown that the easiest way to record information on typing results is to establish an isolate-based database, with each line of information relating to a viral isolation resulting in CPE. Thus, if a single AFP case has two specimens collected and each specimen is inoculated onto two cell lines, and a virus isolate is obtained in each inoculated cell line, there will be four lines of information in the isolate database relating to the AFP cases. Information on the isolates can be linked to information on the specimen through the specimen Id number and the EPId number.
The recommended information to be collected and recorded on typing of virus isolates should include the following:
· EPId number of case;
· laboratory specimen number;
· isolate ID number (a number based on the specimen laboratory number is recommended);
· date of typing;
· typing plate identification;
· typing result: if polio present, the type(s);
— if NPEV present, whether it was typed or not;
— if typing needs to be repeated;
· date final typing results available;
· date results sent to National EPI manager;
· date sent to WHO Regional Office;
· date virus isolate sent to Regional Reference Laboratory;
· date ITD results received from Regional Reference Laboratory.
All poliovirus isolates should be characterized by intratypic differentiation as soon as possible after isolation. Recording information on intratypic differentiation is now complicated because several National Polio Laboratories are capable of carrying out one or more intratypic differentiation tests. The current recommendation is that all poliovirus isolates originating from cases of AFP, or from suspected outbreaks of poliomyelitis, must be confirmed and characterized by intratypic differentiation in a laboratory accredited by WHO to carry out such differentiation. Poliovirus isolates originating from routine clinical virological investigations, epidemiological surveys and environmental studies where wild poliovirus is not expected to occur may be characterized by accredited National Laboratories capable of carrying out intratypic differentiation. Acceptance of the results of these laboratories is at the discretion of the WHO Regional Offices and the Regional Polio Certification Committees.
The minimum recommended information to be sent with the material for intratypic differentiation includes the following:
· case identification number (EPId);
· laboratory specimen number;
· isolate identification number;
· passage history of isolate (e.g. L20B second pass);
· date of sending isolate to RRL.
The minimum recommended information to be recorded by the laboratory carrying out intratypic differentiation includes the following:
· date referred material received;
· EPId number;
· ITD laboratory sample number;
· referring laboratory sample number;
· referring laboratory isolate number;
· material type;
· date of ITD processing by method 1;
· result: polio 1 present (non-Sabin or Sabin-like);
· result: polio 2 present (non-Sabin or Sabin-like);
· result: polio 3 present (non-Sabin or Sabin-like);
· result: NPEV present;
· result: no virus present;
· result: not interpretable;
· date of ITD processing by method 2;
· result: polio 1 present (non-sabin or sabin-like);
· result: polio 2 present (non-sabin or sabin-like);
· result: polio 3 present (non-sabin or sabin-like);
· result: NPEV present;
· result: no virus present;
· result: not interpretable;
· date final ITD result available;
· date result sent to National EPI programme;
· date result sent to national laboratory;
· date result sent to WHO regional office;
· if virus isolates were sent for genomic sequencing:
- date isolates sent;
- sequencing results;
- date sequencing results received.