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Environmental Surveillance of Sewage (ENVS) for polioviruses: with a special
emphasis on detecting prolonged silent circulation.
Lester M. Shulman ([email protected])
For the “Collaborative Silent Polio Circulation Workshop”, Seattle Washington July 1-2,
2015.
Polioviruses can be found in water (surface water, ground water, seawater,
sewage, recreational water, agricultural water and drinking water), on land (soil,
sludge, excretia in non-water sources), and even in the air (powdered sludge,
sewage processing etc). For the purposes of the rest of this discussion ENVS refers
to Sewage surveillance and the examples will describe ENVS for poliovirus. Copies
of our publications describing sustained silent transmission of wild poliovirus in an
Israeli population with vaccine coverage >95% in 2013-2014 have been added as
part of my background material.
Standard procedures identifying viruses and especially poliovirus in
wastewater have not yet been adopted. However the USEPA Manual of Methods for
Virology[1] and WHO Laboratory Manuals for Environmental Surveillance [2] have
[3]been updated to contain is state-of-the-art procedures that have undergone
extensive testing. On of the main goals has been and continues to be optimization of
sample collection and processing.
In 1988 the World Health Assembly endorsed the eradication of poliovirus and
the Global Polio Eradication Initiative was launched. Eradication requires a means for
interrupting transmission of a pathogen and an effective surveillance system to ensure
that interruption has occurred. Two effective vaccines have been used extensively since
the 1950’s, an oral one based on live attenuated strains (OPV) and an i.m. one based on
inactivation of neurovirulent strains. Both are trivalent to raise antibodies against the
three non-cross-reacting serotypes. It is interesting that some of the traits of the live
vaccine that were of advantage to it when poliovirus was endemic are problematic as
the endgame of eradication approaches. The example relevant for ENVS, silent
circulation, and poliomyelitis is the ability of the live vaccine to infect contacts of the
vaccine, mutate, revert to neurovirulent virus, and cause isolated cases of paralytic
poliomyelitis in the vaccinnee or contacts or even outbreaks of poliomyelitis in the
general population.
Acute flaccid paralysis (AFP) surveillance is the isolation and molecular and
serological analysis of all viruses from all cases of AFP, to rule-in or rule-out polioviral
etiology[4]. The rational for AFP surveillance is based on the observation that AFP from
all non-polioviral causes occurs with an incidence of 1 per 100,000 in children up to the
age of 15. When all AFP cases are investigated and the AFP incidence is within the range
for non-polio causes, the absence of poliovirus in the stool samples from any AFP case is
considered to indicate absence of circulating poliovirus in the region under surveillance.
A surveillance area is considered to be wild poliovirus-free when adequate AFP
surveillance levels for greater than 3 years indicate absence of wild poliovirus, and
entire WHO-designated regions are considered to be free from endogenous wild
polioviruses when all countries within that region are wild poliovirus-free. A review of
publications between 1935 and 1995 on excretion of polioviruses by Alexander and
associates [5] indicated that in most infections of naıve children, wild polioviruses were
excreted for 3–4 weeks with a mean rate of 45% at 28 days, and 25% of the cases were
still excreting during the sixth week. In contrast, fewer than 20% excreted vaccine
strains after 5 weeks. Excretion of polio ranged from a few days to several months [6].
The highest probability of detecting poliovirus positive stool samples was reported to
be at 14 days after the onset of paralysis [5, 7] and is the basis of stool sample collection
for diagnosis of polio AFP surveillance. The Global Polio Laboratory Network of
laboratories accredited by the WHO provides the surveillance arm necessary for
successful eradication. Standard laboratory procedures were adopted for acute flaccid
paralysis (AFP) surveillance [4]. ENVS is considered as supplementary surveillance. In
contrast to AFP surveillance, standardized methods for ENVS are still in developmental
stages[2].
OPV strains are present in sewage in countries where OPV is part of the
vaccination program making it much more difficult to screen sewage for the presence of
vaccine-derived viruses and wild viruses in mixtures containing much higher amounts
of vaccine strains. Additional difficulties in detecting polio by ENVS arise from the fact
that shedding may depend in part on immune status and competence of the infected
human host and shedding may be intermittent [8]. Molecular assays offer one of the
best ways to distinguish and even easily quantitate the amount of non-vaccine virus in
these mixtures [9-12].
The following 6 steps are essential for implementing a successful ENVS
program[13, 14]: (1) Identify the target population for the surveillance The optimal
size of the population in the catchment area should be between 100,000 and 300,000,
and sites should be selected for ease of access and lack of contaminants that can inhibit
laboratory molecular and viability studies.; (2) Ensure the necessary resources and
expertise are in place for collection transportation analysis, storage and
decontamination.; (3) Design a practical sampling plan that includes the type of site
(sewage or other), the type of sample (grab (semi-quantitative) vs trap (not
quantitative), the logistics needed for sampling (all lab and non lab needs including
storage and transportation of the sample to the diagnostic lab), the duration and
frequency of sampling (repeated sampling over extended time improves sensitivity and
makes interpretation of negative findings more reliable), and clear lines for
communication); (4) WHO certified sampling processing facilities with appropriately
trained personnel, standard sampling protocols, quality controls, data management
systems, established turn-around-times for virological testing , results and reporting
and appropriate safety standards. SOPs should include rules for storage of the sample
and downstream byproducts and for safe disposal after they are no longer needed; (5)
Detection, interpretation of results, and reporting (SOPs for identification of viruses,
establish turn-around times, and actual reporting of results); and (6) a clear
contingency plan for reacting when non-vaccine polioviruses are detected.
In almost all cases, the amount of poliovirus in sewage is low, and detection
requires a concentration step. Concentration procedures include (1) a two phase
separation after addition of Dextran T40 and PEG 6000 to the sewage (can easily be
implemented in all laboratories of the GPLN); (2) PEG600/NaCl precipitation (requires
heavy centrifuge and rotors not available in all labs); and (3) Tangential Flow
Ultrafiltration (currently being evaluated). Of the 3, the latter may provide the best
solution since early trials indicate much higher viral recovery and it offers the
possibility of on-site use to significantly reduce one of the current bottlenecks of ENVS,
e.g., the need to store and transport large volumes of contaminated sewage under cold
chain conditions).
The WHO standard detection protocol[2] requires viral isolation by tissue
culture. The cell lines used, the number of replicate cultures used for viral challenge,
and the inoculation volumes an even the number of days post-inoculation have not yet
been standardized and these all influence results especially when virus is in low
concentration.
Three examples will be briefly presented to show the usefulness of ENVS in
the GPEI; ENVS in Egypt, in Israel and in Finland/Slovakia/Estonia.
In Egypt, ENVS was initiated in September 2000 while wild poliovirus was
still endemic but in the last stages of elimination. Results in 2001-2002 revealed
widespread silent circulation of wild type 1 poliovirus, helped to identify gaps in
routine AFP surveillance, and was helpful in targeting surveillance and
immunization so that by the end of 2006 Egypt was successful in eliminating
endemic circulation of wild polio. In 2005 and 2010 type 2 aVDPVs were isolated
and wild type 1 polio originating from Pakistan was isolated from sewage in Cairo in
December 2012.
Countrywide routine monthly ENVS was initiated in Israel in 1989 in
response to the last outbreak of poliomyelitis. ENVS identified the introduction,
local silent circulation of wild polio, and documented the successful efforts to
control the circulation with tOPV in the Gaza District between 1995-96[14, 15].
Advanced molecular analysis techniques were developed to distinguish between
multiple introductions of wild poliovirus and local endemic circulation [16]. In 2005
Israel switched to exclusive use of inactivated polio vaccine for routine polio
vaccination and OPV strains disappeared from sewage within a few months.
Between 1998 and 2014, neurovirulent, vaccine derived polioviruses (aVDPVs)
were periodically isolated from sewage from central Israel, once in Jerusalem, and
twice from Haifa. Molecular analysis suggested that the aVDPVs were excreted by at
least three persistently infected immune-deficient individuals ([17] and Shulman
unpublished). After 2005 when sewage was OPV-free, all polio that was isolated was
of potential interest. Plaque assay was used to indicate the amount of virus
recovered from each sewage sample. During this time, the highly sensitive ENVS
used in Israel enabled tracking of the location of the individuals who were excreting
the aVDPVs as they moved from city-to-city and even within a single city [14].
Finally, in 2013, ENVS detected a sharp rise in the number of poliovirus plaques that
analysis confirmed as wild type 1 (WPV1) [18]. Sequence and phylogenetic analysis
revealed that the imported wild type 1 poliovirus originated from Pakistan and was
closely related to the wild type 1 poliovirus isolated in December 2012 in Cairo from
sewage [19]. Poliovirus isolated from AFP cases in Syria in 2014 were also related to
the viruses in Israel and Egypt [20]. Specific RT-PCR primers were designed to
detect this wild type virus by ENVS[12]. More importantly these primers and probe
were validated for use in semi-quantitative analysis of RNA extracted directly from
concentrated sewage allowing an initial turn-around time of a week after sewage
collection [12, 18, 21]. Results were confirmed by standard tissue culture two weeks
later. The results from qRT-PCR were correlated with the number of plaques so that
when the environment became flooded with OPV strains after an SIA with bOPV in
August 2013, plaque-equivalent wild poliovirus loads could continue to be provided
to the Israel Polio Oversight Committee for outbreak control. The ENVS helped
successfully pinpoint the epicenter of silent circulation and target the cohort with
the highest probability of detecting silent circulation by stool survey of identified
individuals [21, 22]. A second stool survey to document the decline in circulation of
WPV1 was conducted after the bOPV SIA. Some of the stools in the second survey
contained type 1 OPV strains. Based on the average PFU of type 1 OPV excreted per
gram of stool, the average weight of stool excreted per day, and a factor
compensating for dilution of virus in the sewage based on quantitative recovery of
virus downstream of a site where it was introduced (Shulman, unpublished) we
were able to estimate the number of individuals excreting vaccine. These numbers
correlated well (r=0.892; p<0.001) with records of the actual numbers of
individuals who were vaccinated each week according to emergency response
weekly immunization records obtained from the Ministry of Health (Perepliotchikov
et al unpublished). Using the average number of PFU of wild type 1 per gram in the
first and stool survey we were able for the first time to estimate the weekly number
of silent WPV1 excretors in different cities. The Regional Certification Committee
(RCC) required that the same sensitive methods that were used to identify the silent
circulation of the WPV be used to document its disappearance for 12 months after
the last positive ENVS sample collected on April 3rd 2014 before Israel could be
declared WPV1-free. As a consequence of very high population immunity to polio
(>95%,), there were no polio-AFP cases during the entire episode (Feb 2013- April
2015).
The Polio Laboratory in Finland recovered highly diverged aVDPVs from
sewage in Finland, Slovakia and Estonia over extended periods of time [13, 23].
Molecular analysis strongly suggested that these viruses were shed by immunedeficient individuals. These findings may indicate that persistent excretors may be
less rare than believed on the basis of identification of known persistent excretors
and failed efforts to identify new ones by screening immune deficient individuals in
multinational studies.
There are a number of challenges for ENVS that need to be addressed. In the
field these include interpretation of data from open sewers where water flow, depth
and contamination and the number of persons in the catchment area are poorly
documented, unknown effects of pooling samples from different locations to reduce
workload, and the high cost and difficulty of transporting large volumes of sewage
within and between countries in time frames that results will be are relevant for
intervention. In the lab they include space personnel and interpretation of data
especially negative results. On-site processing would help to alleviate some of these
difficulties. In the lab they involve the process of concentration, virus concentration,
elution from particles and filters, and reduction of co-extraction of inhibitors.
In conclusion ENVS will be a critical element of surveillance during polio
eradication and in the post eradication era. It will play an important role in polioendemic countries, countries where poliovirus has been reintroduced and in poliofree countries. It is especially helpful in countries where there is sub-optimal AFP
surveillance and high risk for transmission and for countries with very high vaccine
coverage (the example given here is Israel) where there is a significantly reduced
likelihood for development of poliomyelitis after infection with neurovirulent
poliovirus strains and risk of much higher numbers of silent infections by nonvaccine polioviruses before any cases occur. The objective on ENVS for eradication
is to measure disappearance of endemic strains, imported strains, finally live
vaccine strains and to provide early warning of importation or re-emergence. The
sewage from the polio ENVS can be and has been used to identify circulation of
other enteric viruses.
Supplemental reading list for silent circulation of wild type 1 poliovirus in
Israel in 2013-2014.[12, 14, 18, 19, 21, 22, 24-26]
Anis E, Kopel E, Singer SR, Kaliner E, Moerman L, Moran-Gilad J, Sofer D,
Manor Y, Shulman LM, Mendelson E, Gdalevich M, Lev B, Gamzu R,
Grotto I. Insidious reintroduction of wild poliovirus into Israel, 2013. Euro
Surveill 2013; 18(38).
Hindiyeh MY, Moran-Gilad J, Manor Y, Ram D, Shulman LM, Sofer D,
Mendelson E. Development and validation of a real time quantitative
reverse transcription-polymerase chain reaction (qRT-PCR) assay for
investigation of wild poliovirus type 1-South Asian (SOAS) strain
reintroduced into Israel, 2013 to 2014. Euro Surveill 2014; 19(7): 20710.
Kalkowska DA, Duintjer Tebbens RJ, Grotto I, Shulman LM, Anis E, Wassilak
SG, Pallansch MA, Thompson KM. Modeling options to manage type 1
wild poliovirus imported into Israel in 2013. J Infect Dis 2015; 211(11):
1800-1812.
Manor Y, Shulman LM, Kaliner E, Hindiyeh M, Ram D, Sofer D, Moran-Gilad J,
Lev B, Grotto I, Gamzu R, Mendelson E. Intensified environmental
surveillance supporting the response to wild poliovirus type 1 silent
circulation in Israel, 2013. Euro Surveill 2014; 19(7): 20708.
Moran-Gilad J, Mendelson E, Burns CC, Bassal R, Gdalevich M, Sofer D,
Oberste MS, Shulman LM, Kaliner E, Hindiye M, Mor O, Shahar L, Iber J,
Yishay R, Manor J, Lev B, Gamzu R, Grotto I. Field study of fecal
excretion as a decision support tool in response to silent reintroduction of
wild-type poliovirus 1 into Israel. J Clin Virol 2015; 66: 51-55.
Shulman LM, Gavrilin E, Jorba J, Martin J, Burns CC, Manor Y, Moran-Gilad J,
Sofer D, Hindiyeh MY, Gamzu R, Mendelson E, Grotto I. Molecular
epidemiology of silent introduction and sustained transmission of wild
poliovirus type 1, Israel, 2013. Euro Surveill 2014; 19(7): 20709.
Shulman LM, Manor Y, Sofer D, Mendelson E. Bioterrorism and Surveillance for
Infectious Diseases - Lessons from Poliovirus and Enteric Virus
Surveillance J Bioterr Biodef 2012; S4.
Shulman LM, Martin J, Sofer D, Burns CC, Manor Y, Hindiyeh M, Gavrilin E,
Wilton T, Moran-Gilad J, Gamzo R, Mendelson E, Grotto I, Group GPI,
Group GPIG-PI. Genetic analysis and characterization of wild poliovirus
type 1 during sustained transmission in a population with >95% vaccine
coverage, Israel 2013. Clin Infect Dis 2015; 60(7): 1057-1064.
Shulman LM, Mendelson E, Anis E, Bassal R, Gdalevich M, Hindiyeh M, Kaliner
E, Kopel E, Manor Y, Moran-Gilad J, Ram D, Sofer D, Somekh E, Tasher
D, Weil M, Gamzu R, Grotto I. Laboratory challenges in response to silent
introduction and sustained transmission of wild poliovirus type 1 in Israel
during 2013. J Infect Dis 2014; 210 Suppl 1: S304-314.
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Shulman LM, Manor Y, Sofer D, Mendelson E. Bioterrorism and Surveillance
for Infectious Diseases - Lessons from Poliovirus and Enteric Virus
Surveillance J Bioterr Biodef 2012; S4.
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surveillance supporting the response to wild poliovirus type 1 silent
circulation in Israel, 2013. Euro Surveill 2014; 19(7): 20708.
Shulman LM, Gavrilin E, Jorba J, Martin J, Burns CC, Manor Y, Moran-Gilad J,
Sofer D, Hindiyeh MY, Gamzu R, Mendelson E, Grotto I. Molecular
epidemiology of silent introduction and sustained transmission of wild
poliovirus type 1, Israel, 2013. Euro Surveill 2014; 19(7): 20709.
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Shulman LM, Mendelson E, Anis E, Bassal R, Gdalevich M, Hindiyeh M, Kaliner
E, Kopel E, Manor Y, Moran-Gilad J, Ram D, Sofer D, Somekh E, Tasher D, Weil
M, Gamzu R, Grotto I. Laboratory challenges in response to silent
introduction and sustained transmission of wild poliovirus type 1 in Israel
during 2013. J Infect Dis 2014; 210 Suppl 1: S304-314.
Moran-Gilad J, Mendelson E, Burns CC, Bassal R, Gdalevich M, Sofer D,
Oberste MS, Shulman LM, Kaliner E, Hindiye M, Mor O, Shahar L, Iber J, Yishay
R, Manor J, Lev B, Gamzu R, Grotto I. Field study of fecal excretion as a
decision support tool in response to silent reintroduction of wild-type
poliovirus 1 into Israel. J Clin Virol 2015; 66: 51-55.
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Shulman LM, Martin J, Sofer D, Burns CC, Manor Y, Hindiyeh M, Gavrilin E,
Wilton T, Moran-Gilad J, Gamzo R, Mendelson E, Grotto I, Group GPI, Group
GPIG-PI. Genetic analysis and characterization of wild poliovirus type 1
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during sustained transmission in a population with >95% vaccine coverage,
Israel 2013. Clin Infect Dis 2015; 60(7): 1057-1064.
Kalkowska DA, Duintjer Tebbens RJ, Grotto I, Shulman LM, Anis E, Wassilak
SG, Pallansch MA, Thompson KM. Modeling options to manage type 1 wild
poliovirus imported into Israel in 2013. J Infect Dis 2015; 211(11): 18001812.
Anis E, Kopel E, Singer SR, Kaliner E, Moerman L, Moran-Gilad J, Sofer D,
Manor Y, Shulman LM, Mendelson E, Gdalevich M, Lev B, Gamzu R, Grotto I.
Insidious reintroduction of wild poliovirus into Israel, 2013. Euro Surveill
2013; 18(38).