Poliovirus detection in wastewater and stools following an

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Poliovirus detection in wastewater and stools following an Powered By Docstoc
					Printed in Great Britain.                                                                           International Journal of Epidemiology 2003;32:772–777
                                                                                                                                  DOI: 10.1093/ije/dyg185

Poliovirus detection in wastewater and stools
following an immunization campaign in
Havana, Cuba
Pedro Más Lago,1 Howard E Gary Jr,2 Luis Sarmientos Pérez,1 Victor Cáceres,2 Julio Barrios Olivera,1
Rosa Palomera Puentes,1 Marité Bello Corredor,1 Patricia Jímenez,1 Mark A Pallansch3 and
Roberto González Cruz4

                                    Accepted          25 March 2003

                                    Background Recent outbreaks of poliomyelitis caused by vaccine-derived virus have raised
                                               concerns that vaccine-derived poliovirus may continue to circulate after eradication.
                                               In these outbreaks, the virus appears to have replicated for 2 years before

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                                               detection. Early detection is critical for an effective response to these outbreaks.
                                               Although acute flaccid paralysis (AFP) surveillance will remain the standard for
                                               poliovirus detection, wastewater sampling could be a useful supplement. In this
                                               study, we evaluated the sensitivity of wastewater sampling by concurrently collect-
                                               ing stools from children aged 3 years attending two neighbourhood clinics in
                                               Havana, Cuba, and wastewater from the same neighbourhoods.

                                    Methods           Sample collection was begun during the third week after the national immunization
                                                      campaign, continued weekly through the seventh week, and was repeated during
                                                      weeks 15 and 19. Virus detection and titration were performed using both cell
                                                      culture and polymerase chain reaction techniques.

                                    Results           Wastewater sampling was found to be at least as sensitive as stool sampling under
                                                      these conditions. Poliovirus was isolated from children through week 7, sug-
                                                      gesting that viral shedding reached undetectable levels between weeks 8 and 14.
                                                      The last virus-positive wastewater sample was collected during week 15.

                                    Conclusions Wastewater sampling under the conditions studied can be a sensitive supplement
                                                to AFP surveillance. Similar studies under different conditions are needed to
                                                determine the role of wastewater sampling in post-eradication surveillance.

                                    Keywords          Polio, environmental surveillance, wastewater, OPV

The 1988 World Health Assembly of the World Health Organ-                       Since then, the number of reported polio cases has decreased
ization (WHO) passed a resolution committing to the global erad-                from an estimated 350 000 in 125 countries during 1988 to fewer
ication of the poliovirus by the end of the year 2000 (WHA41.28).               than 1000 in 10 countries during 2001.1 As the number of cases
                                                                                decreases, however, we are faced with additional challenges
                                                                                to our systems for detecting circulating poliovirus. Recent out-
1 Instituto de Medicina Tropical Pedro Kouri, Havana, Cuba.                     breaks of poliomyelitis on the islands of Hispaniola,2,3 the
2 Global Immunization Division, Centers for Disease Control and Prevention,     Philippines,4 and Madagascar5 have demonstrated that vaccine-
 Atlanta, GA, USA.                                                              derived polioviruses can circulate in a virulent form. Further-
3 Division of Viral and Rickettsial Diseases, Centers for Disease Control and   more, regional and global certification committees may rely on
 Prevention, Atlanta, GA, USA.                                                  non-standard sources of information such as wastewater
4 Ministerio de Salud Pública, Republic of Cuba Havana, Cuba.
                                                                                sampling in their task of declaring their areas polio-free. These
Correspondence: Howard E Gary Jr, Polio Eradication Branch, MS E05,             factors accentuate the need to maintain surveillance in polio-
Global Immunization Division, Centers for Disease Control and Prevention,       free areas and after discontinuing oral polio vaccine (OPV) use,
Atlanta, GA 30333, USA. E-mail:
                                                                                and to understand the sensitivities of approaches other than
Reprint requests: Dr Pedro Más Lago, Pedro Kouri Institute of Tropical
Medicine, Autopista Novia del Mediodia, Km 6, entre Autopista Nacional y        acute flacid paralysis (AFP) surveillance for detecting poliovirus
Carretera Central, La Lisa, Ciudad Habana, Cuba. E-mail:       in a population.

                                                           POLIOVIRUS DETECTION AFTER AN IMMUNIZATION CAMPAIGN                             773

   Maintaining the ability to detect circulating poliovirus both          by a concurrent stool survey of vaccine recipients following a
before and after certification will pose unique challenges. One           mass vaccination campaign in Havana, Cuba.
challenge will be to sustain the motivation necessary for
intense AFP surveillance. Another will be the sharp decrease
in the sensitivity of AFP surveillance at low infection rates,6
and the lack of sufficient data to fully understand the conditions
that could give rise to extended low-level transmission. Also,            Study locations
the future expansion of the use of inactivated polio vaccine              Samples were collected following the second of the two 1997–
(IPV), which provides limited protection against infection, could         1998 mass immunization campaigns. Four criteria were used to
contribute to reduced surveillance sensitivity. The ability of genetic-   select study locations in Havana. First, to minimize the presence
ally mutated vaccine viruses to replicate for about 2 years               of industrial or similar waste in the wastewater, the location had
before their detection, as in outbreaks in Hispaniola and the             to be a residential neighbourhood. Second, the neighbourhood
Philippines, both areas that had been declared polio-free,                must have a collective wastewater system that served only that
emphasizes the difficulty and importance of sensitive post-               neighbourhood and have at least two accessible sampling points.
eradication surveillance.                                                 Third, the wastewater at the sampling sites must have come only
   One approach to increasing sensitivity is to supplement AFP            from the target neighbourhood. Finally, at least one polyclinic
surveillance with methods such as sampling wastewater to test             must have been present in the neighbourhood, serving only
for the presence of poliovirus. To use wastewater sampling as an          residents of that neighbourhood. The latter two criteria insure
effective surveillance supplement, we must understand its popu-           that the stool and wastewater samples represent the same

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lation sensitivity, defined as the probability of obtaining at least      population and only that population. We selected two Havana
one sample that is positive for poliovirus when the virus is cir-         neighbourhoods that met these criteria, designated ‘A’ and ‘B’
culating within the population.6 A number of sensitive laboratory         in this report.
procedures have been developed to recover virus from a
sample, but few published data exist to address the sensitivity           Stool sampling
of a wastewater-based surveillance system in a population. Data           Stool samples were collected from randomly selected children
from Sweden7,8 and Finland9 have documented the ability to                aged 3 years attending the neighbourhood polyclinics either
detect vaccine-derived poliovirus in wastewater during periods            for a well child visit or injury. This age criterion captured children
of routine immunization, during wild virus outbreaks, and                 targeted for vaccination in the second round of the mass cam-
following a national campaign. Researchers in Finland recently            paign. Because the study’s main objective was to compare rates
demonstrated the ability to detect vaccine virus in a municipal           of viral shedding with rates of viral detection in wastewater, we
wastewater collection system for 4 days after introducing a               only recorded each child’s date of birth and place of residence.
known quantity of vaccine virus into the system.10 These data             No additional information on medical history or vaccine status
provide only limited ability to compare the results from waste-           was collected. However, Cuba has a long history of maintaining
water sampling to other measures or indicators of virus circulation.      very high participation rates in its National Immunization Days
   Fewer studies have attempted to relate the rate of virus shed-         (NID).16 We assumed, therefore, that almost all of the children
ding within the population to the likelihood of detecting virus           in this study had received OPV prior to stool collection.
in wastewater. One such study was conducted as part of an                    Beginning in the third week after the last national vaccination
immunization campaign using Sabin type 3 OPV during a 1960                campaign (week 3), samples were collected through week 7,
type 3 outbreak in Atlanta, GA.11 Periodic wastewater samples             with additional samples collected during weeks 15 and 19
were collected from neighbourhoods recently the targets of                through 21. (Results for the weeks 19–21 samples were grouped
a type 3 OPV immunization campaign, while concurrent stool                and are reported here as week 19 results.) Our target was to
samples were collected from children attending daycare within             collect stools from 50 children in each neighbourhood during
the same neighbourhoods. The stool samples yielded virus for              each sampling week. Children were enrolled in the study after
12 weeks following the campaign, while the wastewater samples             we obtained informed consent from one of the parents. The parent
yielded virus for 14 weeks. Although these data provide useful            was given a container and instructions for stool collection. The
information, they were collected as part of an immunization               parent kept the stool sample refrigerated until returning it to the
campaign in communities chosen because of their risk status,              family physician at the polyclinic. The samples were then taken
not because of their suitability for studying wastewater sampling.        from the clinic to the Instituto Pedro Kouri (IPK) for testing.
In addition, the samples were processed and tested by simpler,
less sensitive methods than those currently available.                    Wastewater sampling
   Cuba provides an ideal environment in which to investigate             The methods we used for collecting and testing wastewater
the sensitivity of wastewater sampling. Since the early 1960s,            samples were chosen over potentially more sensitive methods
Cuban health officials have administered OPV solely through               because they are more easily implemented in a wide variety
mass immunization campaigns;12–15 no OPV is given through                 of laboratories (WHO field guide in preparation). Wastewater
routine childhood immunization. Because of this, it is possible           samples were collected from two separate locations within each
to estimate the proportion of recipients shedding virus starting          neighbourhood during the same weeks in which stools were
from a known time of introduction, without background virus               collected. During each sampling week, samples were collected
from routine immunization. In the study described here we com-            on two different days from each of the sampling sites. On each
pared the frequencies of poliovirus detections in wastewater              of these days, 500 ml were collected from each site in the morn-
with the rates of viral shedding in the community as estimated            ing and again in the afternoon. These were combined into a

single 1-l composite sample for each site. Samples were collected        For the PCR procedures, RNA extraction was performed with
by dipping the collection flask directly into the wastewater          TRIZOL (Life technologies TM, Gibco BRL) according to the
effluent. The outer surface of the flask was then rinsed and the      manufacturer’s instructions. Briefly, 0.25 ml of concentrated
flask placed in a cold box for transport to IPK.                      sewage samples were homogenized in 0.75 ml of TRIZOL and
                                                                      0.2 ml of chloroform. After centrifugation at 4°C for 15 minutes
Isolation and identification of poliovirus in stool                   at 12 000 × g, the aqueous phase containing RNA was precipitated
Virus isolation was performed according to the following methods.     with isopropanol (v/v) at –20°C overnight. After centrifugation
First, for each stool specimen a 20% (w/v) suspension was made        for 10 minutes at 16 000 × g the supernatant was discarded and
using phosphate buffered saline (PBS) with antibiotics. Chloro-       the pellet washed in l ml of 75% ethanol, dried and suspended
form was then added to the mixture, agitated vigorously, and          in 50 µl of diethylpyrocarbonate (DEPC)-treated water.
the mixture clarified by centrifugation at 10 000 r.p.m. in an           Sequences of a primer pair matching highly conserved
Eppendorf centrifuge. The supernatant was kept frozen at –20°C        sites within the 5’-non-coding regions of enterovirus genomes
until innoculation onto cell monolayers. For innoculation, 0.2 ml     were EV PCR-1 (A, 539 to 565) 5’ACACGGACACCCAAAGTA-
of the supernatant was innoculated on rhabdomyosarcoma                GTCGGTTCC-3’, and EV PCR-2 (S, 452 to 476) 5’-TCCGGCCC-
(RD) and L20b cell lines. The culture tubes were then incubated       CTGAATGCGGCTAATCC-3’.
at 37°C and examined every 24 hours for evidence of cytopathic           The degenerate primers used for amplifying poliovirus sequences
effect (CPE). Samples showing CPE were frozen and thawed for          were pan PV PCR-1 (A, 2935 to 2954) 5’-TTIAIIGC(A/G)TGICC-
passage onto new cells. A sample was observed for 12 days before      (A/G)TT(A/G)TT-3’, and pan PV PCR-2 (S, 2875 to 2894)
being considered negative. For samples showing CPE in L20b            5’-CITAITCI(A/C)GITT(C/T)GA(C/T)ATG-3’. The number in

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cells, poliovirus serotypes were identified by neutralization with    parentheses indicates the genomic intervals matching the pri-
hyperimmune serum pools. Four preparations were formed by             mers (A, antisense or antigenome polarity; S, sense or genome
mixing sera with antibodies against types 1 and 2, types 1 and        polarity), according to the numbering system of Toyoda et al.17
3, types 2 and 3, or types 1, 2, and 3 in a solution containing       Deoxyinosine residues are indicated by the letter I. Primer
50 neutralization units of each type of antiserum and approxi-        positions having equimolar amounts of two different nucleo-
mately 100 TCD50 of the isolate (32–320 TCD50). Because the           tides are enclosed in parentheses.
L20b cells are less sensitive than the RD cells, all samples show-       In vitro amplification by PCR was performed by a modification
ing CPE only in RD cells were passed onto L20b cells also. If CPE     of methods described previously.18 Amplification reactions were
resulted in the L20b cells, the identification proceeded as previ-    carried out in 50 µl reaction mixture containing RNA template
ously described.                                                      in PCR buffer (67 mM Tris-HCL; pH 8.8, 17 mM NH4SO4, 6 µM
                                                                      EDTA, 2 mM MgCl2, 1 mM 2-mercaptoethanol), 100 ng of each
Extraction of virus particles from sewage samples                     primer, 100 µM (each) dATP, dGTP, dGTP, dTTP (Pharmacia Bio-
Virus was extracted and concentrated by the method recommended        tech, Piscataway, NJ), 5 U of placental RNase inhibitor (Boehringer
by WHO. The sewage was centrifuged at 4°C for 30 minutes at           Mannheim Biochemicals, Indianapolis, IN.), 1.5 U of avian myelo-
5000 × g to sediment (pellet) the sewage solids. The supernatant      blastosis virus reverse transcriptase (Boehringer Mannheim),
was recovered for further processing. The solids were then re-        and 1.25 U of Taq DNA polymerase (Boehringer Mannheim). The
suspended in 5 volumes of elution medium (3% beef extract),           reaction mixture were overlaid with mineral oil, and incubated
and 1⁄ 2 volume of chloroform added. This mixture was centri-         at 42°C for 30 minutes prior to 30 cycles of programmed ampli-
fuged at 4°C for 30 minutes at 5000 × g to sediment the               fication (denaturation at 94°C for 45 sec, annealing at 42°C
chloroform and sewage solids, and the aqueous supernatant was         for 45 seconds, and extension at 70°C for 45 seconds) in a DNA
recovered. The chloroform and sewage solids were supplemented         thermal cycler (Perkin-Elmer-Cetus). Conditions for polyacrylamide
with another volume of elution medium, the mixture re-                gel electrophoresis and detection of amplified products by
extracted and centrifuged, and the resulting aqueous supernatant      ethidium bromide staining were as described previously.19
recovered. The supernatant extracts from the sewage solids were          Equal numbers of test samples and negative controls were
combined with the original sewage supernatant and viruses were        processed in each test batch to detect possible extraneous nucleic
precipitated from the mixture with polyethylene glycol 8% (PEG)       acid sequences contaminating the RNA extraction and poly-
and sodium chloride 0.3 M (NaCl) at 4°C overnight. The precipi-       merase chain reaction (PCR) reagents were amplified by PCR.
tated viruses were sedimented by centrifugation at 5000 × g for       Additional procedures to avoid PCR contamination were adopted
1 hour at 4°C. The resulting sediment was re-suspended in 2 ml        as elsewhere.20
of PBS and the mixture was extracted with chloroform. After              For virus found in wastewater, serotype identification was
centrifuging to remove the chloroform, the aqueous supernatant        performed using hyperimmune sera and serotype-specific PCR
was recovered and the remaining chloroform was re-extracted           primers.
with a volume of elution medium. After centrifugation, this
elution medium was recovered and combined with the previously
collected aqueous supernatant to obtain the concentrated virus in
a final volume of 6 ml. The sample was then treated with penicillin   Stool samples
(1000 units/ml) and streptomycin (200 µg/ml). A sample was            Stools were collected from 677 children ranging in age from
considered negative if no virus was detected in the undiluted         0 through 35 months. The percentage of children positive for at
concentrate using PCR or cell innoculation methods. The amount        least one poliovirus serotype remained relatively constant from
of virus in the positive samples was titrated using serial dilution   week 3 through week 6, and then declined beginning week 7.
with virus detection performed using both PCR and virus isolation.    None was found to be positive during weeks 15 or 19 (Table 1).
                                                                 POLIOVIRUS DETECTION AFTER AN IMMUNIZATION CAMPAIGN                                  775

Table 1 Percentages of children shedding poliovirus by virus serotype and week after the immunization campaign

                             Any serotype                        Type 1                 Type 2                Type 3                  NPEVa
                   No.           No.          %         upper        No.          %         No.          %        No.            %        No.            %
Week            tested       positive   positive         limit   positive   positive    positive   positive   positive     positive   positive     positive
3                  100             26      26%           32%          10       10%            6        6%          15         15%          18         18%
4                   93             22      24%           30%           7        8%            5        5%          15         16%          21         23%
5                  102             31      30%           37%          15       15%           11       11%              8       8%          19         19%
6                   94             25      27%           33%          18       19%            1        1%              9      10%          21         22%
7                   90             12      13%           20%           5        6%            5        6%              6       7%          47         52%
15                 105              0       0%            2%           0        0%            0        0%              0       0%          56         53%
19                  93              0       0%            2%           0        0%            0        0%              0       0%          44         47%
Total              677           116       17%                        55        8%           28        4%          53          8%         226         33%
a Non-polio enteroviruses.

Roughly the same pattern was observed for each of the three                      containing virus declined after that; the last positive sample was

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individual serotypes. To provide a more conservative point                       collected during week 15. Only one positive sample was found
of comparison for determining the sensitivity of wastewater                      for this week, and it was found to be positive by both virus
sampling, we also calculated the 90% upper confidence limit                      isolation and PCR.
for each week’s percentage of children shedding. The lowest                         Comparing virus isolation with PCR, we found that the per-
upper limit was 2% during weeks 15 and 19. The upper bound                       centage of samples positive by PCR was equal to or greater than
after combining these two weeks was 1%.                                          the percentage positive by virus isolation during each week.
   The pattern for non-polio enteroviruses (NPEV), however,                      However, we did not find virus in any of the week 19 samples
was opposite that for polioviruses. The percentage of samples                    using either method.
positive for NPEV increased sharply during week 7, and remained                     We also determined the virus titre for each positive sample.
at this level during weeks 15 and 19. During each week, the                      From week 6 forward, virus was detected only in the undiluted
percentage of children positive for poliovirus decreased with                    sample by either detection method, so the virus titre added
age. Combining all time periods, 41% (20/49) of the children                     no additional information. Examining the mean log virus titres
under 6 months of age were positive for poliovirus, 22%                          from weeks 3 to 5 reveals a different pattern, however. For
(28/130) of those 6–11 months of age, 16% (36/231) of those                      weeks 3 and 4, all of the samples contained poliovirus, but
12–23 months of age, and 12% (31/258) of those 24–35 months                      the geometric mean viral titre increased sharply between these
of age.                                                                          weeks. During week 3, the geometric mean viral titres were
                                                                                 1:7.5 by both virus isolation and PCR. By week 4, however,
Wastewater samples                                                               the titres were 1:177.8 and 1:74.0 by isolation and PCR respect-
By either virus isolation or PCR, the percentage of wastewater                   ively. By week 5, only 5 of the 8 samples collected were positive
samples positive for poliovirus declined with time (Table 2).                    for poliovirus. The geometric mean viral titers for these positive
During weeks 3 and 4 after the campaign, all samples were found                  samples were 1:4.0 by using virus isolation and 1:10.0 by using
to contain virus regardless of testing method. The percentage                    PCR.

Table 2 Percentages of wastewater samples containing poliovirus by virus serotype and week after the immunization campaign

               Any serotype                                                             Any serotype
               (virus isolation)                    (pan-polio PCRa)                    Type 1                Type 2                  Type 3
                   No.           %        Mean          No.            %      Mean          No.          %        No.            %        No.            %
Week           positive   positiveb     log titre   positive     positive   log titre   positive   positive   positive     positive   positive     positive
3                     8       100%        0.875             8     100%        0.875           4       50%              3      38%              2      25%
4                     8       100%        2.250             8     100%        1.875           7       88%              0       0%              4      50%
5                     5         63%       0.600             5       63%       1.000           3       38%              1      13%              3      38%
6                     1         13%            –            3       38%            –          0        0%              1      13%              0       0%
7                     0            0%          –            3       38%            –          0        0%              0       0%              0       0%
15                    1         13%            –            1       13%            –          1       13%              0       0%              0       0%
19                    0            0%                       0        0%                       0        0%              0       0%              0       0%
Total               23          41%                        28       50%                      15       27%              5       9%              9      16%
a Polymerase chain reaction.
b Eight samples were collected for testing each week.

Relationship of wastewater isolation to rate                           world in which many infants will not be represented in a waste-
of viral shedding                                                      water sample. The absence of these children from our samples
To compare the percentage of children shedding virus with the          then, does not compromise our objective of evaluating this
probability of a positive wastewater sample, we fitted a probit        method for virus detection.
cubit curve to the weekly wastewater results using the propor-            We found it interesting that the levels of type 2 poliovirus
tion of children shedding poliovirus as the independent variable       were low both in stool and in wastewater. Generally, type 2
using SAS Proc Probit {SAS, 1999 #1561}. Using the best fit-           Sabin virus produces a greater immunological response than
curve (intercept = 1.72, slope = –7.76), we estimated that a           the other serotypes, especially after the first dose. Almost all of
shedding rate of 31% is required to have a 0.75 probability of         the children who participated in the second round of NID had
obtaining a positive wastewater sample.                                already received at least one dose of trivalent OPV, so it is likely
                                                                       that most had responded well to the type 2 component of earlier
Serotype-specific results                                              doses, and were less likely to shed virus. Unfortunately, we have
Overall, type 1 was found most often (8% of stools; 27% of waste-      no data from this study to either support or refute this hypothesis.
water samples), followed by type 3 (8% and 16%), with type 2              We also found it interesting that in the stool specimens, the
found least often (4% and 9%). Comparing serotype-specific             rates of polio and non-polio enterovirus isolations were inversely
isolations from stool by week, we found that the percentage of         related. The pattern was found across all age groups (data not
children shedding type 1 virus peaks during weeks 5 (15%) and          presented), suggesting that the OPV virus may have interfered
6 (19%), dropping to only 6% during week 7. The percentage             with NPEV in the gut or in the cell culture. Unfortunately, we
shedding type 2 virus is 6% or lower for all but week 5, during        do not have data that would allow us to investigate this obser-

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which 11% where shedding type 2. The pattern for type 3 shed-          vation further.
ding was slightly different. During weeks 3 and 4, type 3 was             Our findings about the sensitivity of wastewater sampling are
the most frequently isolated serotype (15% and 16% of chil-            encouraging, but we must be cautious about generalizing too
dren tested, respectively). Also, the shedding pattern for type 3      broadly. The first caveat is that wastewater collection systems
did not have a distinct peak as was seen for the other serotypes.      differ widely among countries and communities, and sensitivities
   The serotype-specific temporal patterns for virus isolation         for detecting poliovirus may differ considerably among these
from wastewater differed from the stool isolation patterns. In         systems. In the neighbourhoods we sampled in Havana, waste-
wastewater, isolation of each of the serotypes showed a distinct       water is carried away from the houses by an inter-connected,
peak. The highest isolation rate for types 1 and 3 where during        closed system that empties into an open canal. The areas were
week 4 (88% and 50%, respectively). The highest rate for type 2,       specifically chosen to be free of industrial waste or agricultural
however, was during week 3 (38%). During all weeks but                 runoff. Other methods that are used to dispose of wastewater
week 6, the percentage of samples positive for type 1 was equal        include collective systems that carry the waste to a central treat-
to or higher than the percentages for types 2 or 3. During week        ment facility, open channels that lead from houses or com-
6, one sample was found to contain a type 2 virus, while neither       munities to larger open canals, septic tanks, and pit latrines. The
of the other serotypes was found. The last sample found to contain     sensitivities of sampling from these different systems have not
virus was from week 15; this sample contained type 1 virus.            been systematically compared. The second caveat is that differ-
                                                                       ent laboratory processing and detection methods may have
                                                                       different detection sensitivities. For example, the data from our
Discussion                                                             study indicate that detection by using PCR may have been more
In this study we compared the frequencies of viral detection           sensitive than detection using cell culture.
in wastewater with concurrent estimates of the proportions of             From the standpoint of the polio eradication programme, an
children shedding poliovirus. The results indicate that sampling       important question is whether wastewater sampling can become
wastewater from a collective system as is found in Havana can          a standard tool to supplement AFP surveillance. If this is to
be a sensitive procedure for detecting vaccine-derived poliovirus      happen, we must have available a robust procedure that can be
in a population. The results also show that PCR can be at least        implemented in a wide variety of laboratories. Our finding that
as sensitive for detecting poliovirus as the use of cell culture and   the technically simpler cell culture method produced results
hyperimmune sera. Finally, this study supports earlier findings        similar to those using PCR is encouraging. Additional systematic
that vaccine virus is present in the Cuban population for only a       comparisons of methods with different types of samples are still
few weeks following a mass campaign.21,22                              needed.
   We were able to detect type 1 virus in the wastewater when             The third caveat is that the sensitivity of any detection system
only about 6% of the sampled children were shedding this sero-         can be increased by collecting and testing more samples. Because
type. Furthermore, we found virus in wastewater samples in             of this, evaluation of candidate supplemental surveillance ap-
the absence of positive stool 15 weeks following the mass cam-         proaches should also compare the amount of effort and resources
paign. At the other extreme, all of the wastewater samples tested      each would require to achieve equal sensitivities. During the
positive for at least one virus serotype when about 25% of the         first week of sampling in our study, for example, 26% of the
sampled children were shedding. These findings indicate that           children were shedding at least one of the poliovirus serotypes,
wastewater sampling under these conditions can be a sensitive          while 100% of the wastewater samples were positive. With this
tool for moderately low levels of poliovirus circulation.              level of shedding, testing a single stool sample would have a
   The wastewater samples from this study may not reflect virus        sensitivity of 26%, while testing a single wastewater sample would
excretion from children wearing diapers. We assume, however,           have a sensitivity near 100%. Under these same conditions, how-
that Havana is similar to a large number of cities throughout the      ever, a random sample of 16 children would raise the sensitivity
                                                              POLIOVIRUS DETECTION AFTER AN IMMUNIZATION CAMPAIGN                                   777

of the stool survey to more that 99% (i.e. a probability 0.99                 we must identify which types of wastewater collection systems
of detecting at least one poliovirus).                                        are reasonable to sample, which processing and testing methods
   As a final caveat, it must be understood that we can learn                 are feasible and cost-effective for more widespread use, and
nothing about the transmission or circulation of vaccine-derived              what sampling schemes can yield the best results. Only after we
polioviruses from these data. The patterns of poliovirus isolations           obtain a better understanding of the limits of wastewater-based
could reflect decreasing levels of circulation, latency within the            surveillance will we be able to determine its appropriate role in
wastewater system, the normal range of viral shedding durations,              the final stages of polio eradication.
or all of these.
   In conclusion, this study has provided important data that
have expanded our understanding of the sensitivity of waste-                  Acknowledgements
water sampling and testing. Before we attempt to implement                    The research reported here was funded by the Pan American
this approach more widely, we need additional studies and data                Health Organization. The authors wish to acknowledge the
to estimate the sensitivity of these methods for early detection              invaluable assistance of Dr Ciro de Quadros, Dr Claudio Silviera,
of an outbreak. We especially need more data that inform us of                and Ms Nina Toro of PAHO; Dr Guadalupe Guzman and
the sensitivity of wastewater sampling relative to AFP surveil-               Dr Alina Llop of the Instituto Pedro Kouri; and, Ms Yvonne
lance at different levels of circulation. As part of this work,               Stifel of the Centers for Disease Control and Prevention.

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    •   Wastewater surveillance can be a sensitive supplement to acute flaccid paralysis surveillance at moderate levels
        of poliovirus circulation when sampling from a collective sewage system.

    •   Virus detection using polymerase chain reaction proved to be as sensitive for poliovirus detection as methods
        based on cell culture and neutralization.

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