Poliovirus surveillance by examining sewage specimens. Quantitative recovery of virus after introduction into sewerage at remote upstream location.

In order to assess the feasibility of environmental poliovirus surveillance, known amounts of poliovirus type 1, strain Sabin, were flushed into the sewage network of Helsinki. Grab specimens collected at a remote downstream location and concentrated about a 100-fold revealed infectious poliovirus on four successive days in all three separate experiments. As for concentration, a simple two-phase separation method was found to be at least as useful as a several-fold more resource-demanding polyethylene glycol (PEG) precipitation method. Recovery of the introduced virus was remarkably high (more than 10%). Using the current system, it might be possible to detect poliovirus circulation in a population of 700,000 people by examining a single 400 ml sewage specimen, if 1 out of 10,000 inhabitants were excreting the virus. It is concluded that environmental surveillance is a sensitive approach to monitor silent poliovirus circulation in populations served by a sewage network.

Age- and dose-interval-dependent antibody responses to inactivated poliovirus vaccine.

Antibody responses were studied in five groups of children immunized with different three-dose schedules of inactivated poliovirus vaccine (IPV). The age of the child at the first dose (1-4 months) and the interval between the first and second doses (2-4 months) influenced the initial responses in a serotype-dependent manner. All the groups attained sufficient antibody level after three doses but the third dose given at 18 months resulted in higher persisting antibody levels than that given at 12 months. The highest persisting antibody titers against PV1 and PV2 (but not against PV3) at the age of 3 years were measured in the control group immunized with the current schedule (P < 0.001) in which the first dose is given at 6 months. The level of maternal antibodies present at the time of the first dose correlated negatively with the antibody titers as late as at 3 years of age. It is concluded that three doses of IPV given in widely variable schedules may result in satisfactory immune responses in children but, for optimal results, very early onset of the program and short dosage intervals should be avoided.

Poliovirus-specific intestinal antibody responses coincide with decline of poliovirus excretion.

Antibody responses to poliovirus type 3 were studied in fecal samples of 66 children immunized with 3 doses of enhanced-potency inactivated poliovirus vaccine (E-IPV), followed by 1 dose of monovalent oral poliovirus vaccine (OPV, type 3 Sabin). One fecal sample taken before OPV vaccination and 9 collected thereafter were tested for neutralizing antibodies by a microneutralization assay and for class-specific responses by heavy chain-capture radioimmunoassays. Both neutralizing antibody and IgA responses usually occurred during the second week and coincided with ceasing of virus excretion or a decrease in the excreted virus titer. Half of the vaccinees had received a trypsin-modified E-IPV, but their responses did not differ from those of children immunized with the regular E-IPV. These results are in agreement with the view that an intestinal antibody response, mainly consisting of IgA, may be involved in the ceasing of a primary poliovirus excretion.

Surveillance of patients with acute flaccid paralysis in Finland: report of a pilot study.

WHO recommends that surveillance of patients with acute flaccid paralysis (AFP) be used to demonstrate the eradication of wild poliovirus. In this article we report the results of a study to assess the frequency of AFP patients referred to Finnish hospitals and whether virological diagnostic coverage could be improved by repeated reminders and active feedback. For this purpose, we sent monthly questionnaires to all neurological and paediatric neurological units in Finland, requesting retrospective reporting on investigated paralytic patients with defined clinically relevant diagnoses, rather than AFP. Reminder letters included a pre-paid return envelope. Virological investigations were offered cost free. Of the 492 reporting forms sent, 415 (84%) were returned, evenly covering both the population and the study period (July 1997 to June 1998). Of the 90 patients reported, 83 were evaluable. The apparent incidences of the diagnoses covered were 1.6 per 100,000 at any age, and 1.0 per 100,000 for under--15-year-olds. Guillain-Barré syndrome was the most common diagnosis (0.80 per 100,000). The two faecal specimens required were virologically investigated in nine out of the 10 patients under 15 years of age, but in only 46% of all patients. Four adenovirus strains, but no polioviruses or other enteroviruses, were isolated. We conclude that a satisfactory monthly reporting system was readily established and that a sufficient number of patients with diagnoses resembling AFP are being referred to Finnish hospitals. Active feedback did not increase the proportion of virologically investigated patients to an acceptable level in all age groups. It is clear that other approaches must be used to quantify the circulation of poliovirus in Finland.

Efficacy of PCR and other diagnostic methods for the detection of respiratory adenoviral infections.

Five methods were evaluated for the detection of adenovirus directly from nasopharyngeal aspirates (NPA), including conventional and rapid virus culture, two antigen detection tests, and the polymerase chain reaction (PCR). NPA specimens were obtained from 269 military conscripts suffering from an acute respiratory infection during an adenovirus outbreak. In 133 cases, paired blood specimens were also available. Virus culture followed by a hexon-specific immunofluorescence revealed 159 (59%) adenovirus-positive specimens and it was used as a reference method. In comparison to conventional culture, a rapid 2-day culture method had a sensitivity of 71%. The sensitivities of an enzyme immunoassay and time-resolved fluoroimmunoassay were 53% and 46%, respectively. The PCR method employing Ad7 hexon-specific primers showed a high sensitivity of 94%, and revealed an additional 15 (6%) specimens that could not be confirmed by virus culture. Serology based on significant adenovirus antibody rises had a diagnostic efficacy nearly equal to the virus culture and PCR methods, but a relatively high number of discordant results was found. The present study demonstrates that PCR is a very sensitive rapid diagnostic method for detecting adenovirus specific DNA in NPA specimens of adults.

A population-based prospective survey of newborn infants with suspected systemic infection: occurrence of sporadic enterovirus and adenovirus infections.

BACKGROUND: Enterovirus outbreaks are known to occur in neonatal wards and enteroviruses may cause community-acquired sepsis-like disease in the neonatal period. Less well is known their possible role in suspected systemic infections during the perinatal period. OBJECTIVES: To investigate the occurrence of enterovirus infections in neonatal patients suspected of systemic infection. STUDY DESIGN: A population-based prospective survey was organized in the hospitals of the Greater Helsinki Region during 13 months in 1993-94. Criteria for enrollment included onset of symptoms before the age of 29 days and a decision, on clinical grounds, to take a blood culture for bacteria. Acute phase samples of blood, feces, nasopharyngeal swab, and cerebrospinal fluid, if available, were inoculated in monolayer cultures of four different cell lines. In addition, enterovirus infections were searched for using an enterovirus group-reacting IgM test. RESULTS: One hundred and thirty-seven patients had a sufficient number of specimens examined, and were thus evaluable. Most of the infants had the onset of the symptoms within a few days after birth. An enterovirus was isolated from four newborn infants (3%), while seven children (5%) were found to excrete adenovirus. Enteroviral antigen was detected in cell cultures inoculated with specimens from two additional infants. Virus-positive infants had no evidence of bacterial infection and did not show specific clinical signs or symptoms differentiating them from the rest of the study group. All enrolled infants recovered without sequelae. CONCLUSION: We conclude that sporadic viral infections may be common in neonatal patients with suspected systemic infection, and this should be taken into account when judging the etiology.

A Sabin vaccine-derived field isolate of poliovirus type 1 displaying aberrant phenotypic and genetic features, including a deletion in antigenic site 1.

Poliovirus strains derived from the oral poliovirus vaccine (Sabin) can be differentiated from wild-type poliovirus by tests based on either immunological or genetic properties of the strains. The characterization of a recently identified poliovirus type 1 isolate with exceptional properties is described. Initial phenotypic analysis of the virus by use of polyclonal absorbed antisera suggested a wild-type character. However, the different genomic analyses all confirmed the Sabin-derived character of the virus. All 17 plaques isolated from the strain shared these properties, thus excluding the possibility of a mixture of a wild-type and a Sabin-derived strain. To elucidate the properties of this virus further, the nucleotide sequences of the P1 region and most of the 5' non-coding region were established. Although the nucleotide identity with Sabin 1 was more than 99.4%, mutations were observed in regions encoding three major antigenic sites; the deduced amino acid substitutions confirmed the aberrant results of micro-neutralization assays with site-specific monoclonal antibodies. The most striking feature was the existence of a hexanucleotide deletion in the VP1 gene, which gave rise to a two amino acid deletion in the BC loop. In spite of these antigenic changes, the strain was readily serotyped as poliovirus type 1 under standard conditions. Likewise, replication of the virus under cell culture conditions was not affected by these mutations or by the deletion. Standard polio vaccination protects against this aberrant virus, and its epidemiological significance remains open.

Randomised, controlled trial with the trypsin-modified inactivated poliovirus vaccine: assessment of intestinal immunity with live challenge virus.

The enhanced potency inactivated poliovirus vaccine (E-IPV) was modified to contain trypsin-treated type 3 poliovirus (PV3), strain Saukett, as the type 3 component (TryIPV). This pilot vaccine was previously shown to redistribute the vaccine-induced antibody specificities in mice to mimic those seen in man after poliovirus infection. Groups of infants were then immunised with three doses of TryIPV or E-IPV in a randomised, double-blind trial. Six months after the third dose, at the age of 18 months, the children were challenged with one dose of oral monovalent type 3 poliovirus vaccine. Intestinal immunity was evaluated by assessing the length and extent of PV3 excretion through determination of PV3 titres in 9 successive faecal specimens (2-42 days after challenge). No significant difference in the length or extent of virus excretion was seen between the groups. The results indicate that TryIPV, under the conditions used, was no more potent than the regular E-IPV in inducing resistance to intestinal poliovirus infection.

Randomized comparison of early and late vaccination with inactivated poliovirus vaccine after allogeneic BMT.

Forty-five adult HLA-matched sibling BMT recipients were randomized to receive inactivated poliovirus vaccine (IPV) at 6, 8 and 14 months (early group, n = 23) or at 18, 20 and 26 months after BMT (late group, n = 22). Ninety-five percent of the early group patients had protective antibody titres of > or = 4 to poliovirus type 1 (PV1), poliovirus type 2 (PV2) and poliovirus type 3 (PV3) by a microneutralization assay prior to the first vaccination, at 6 months after BMT. The corresponding proportion for the late group patients was only 67% at 18 months. The antibody responses 1 month after each of the three IPV doses were similar in the two vaccination groups, except that four-fold responses occurred more frequently after the first dose to PV2 and PV3 in the late group. All patients had a protective antibody titre to all poliovirus serotypes 1 and 22 months after the third vaccine dose, except one patient in the early group who lacked antibodies to PV3 at 22 months. Acute GVHD accelerated the decrease of poliovirus antibody titres prior to vaccination but had no influence on vaccination response. Chronic GVHD neither influenced the patient's ability to retain poliovirus antibodies prior to vaccination nor impaired responses to vaccinations. A vaccination schedule consisting of three IPV doses was equally immunogenic when started at 6 or 18 months after allogeneic BMT.

Loss of protective immunity to polio, diphtheria and Haemophilus influenzae type b after allogeneic bone marrow transplantation.

Immunity to poliovirus, diphtheria and Haemophilus influenzae type b (Hib) was studied in 16 adult recipients of a bone marrow transplant from an HLA-identical sibling donor in order to evaluate the need for revaccinations. T-cell depletion was not done in any case. The donors and patients were studied before bone marrow transplantation (BMT) and the patients 1, 3, 6, and 12 months later. Prior to the BMT 10 of 11 patients were immune (titre > or = 4) to all vaccine poliovirus types by a standard microneutralization assay. At 12 months after BMT only two of seven patients were immune to all vaccine types, and none had immunity against an antigenically altered poliovirus type 3 strain Finland. The geometric means of antibody titres against poliovirus types 1, 2, and 3 strain Saukett and strain Finland declined gradually after 1 month postgrafting, being 4.4, 5.4, 3.3, and 1.3 respectively at 12 months after BMT. At 1 year 6 of 11 patients had immunity against diphtheria by a toxin neutralization method, but the antitoxin geometric mean level had decreased to a barely protective level, 0.01 IU/ml. The geometric mean Hib antibody concentration decreased during the first 6 months after BMT and thereafter increased slightly. A significant proportion of BMT recipients lose their protection against polio, diphtheria and Hib, and revaccinations are necessary.

Relative abundance of enterovirus serotypes in sewage differs from that in patients: clinical and epidemiological implications.

One thousand one hundred and sixty-one non-polio enterovirus strains, isolated during regular screening of Finnish sewage specimens, were analysed for serotype distribution seasonally through 20 years, and the findings were compared with similar data based on 1681 clinical isolates. Coxsackievirus B4 (CBV-4), CBV-5, echovirus 11 (EV-11), EV-6, CBV-2 and CBV-3 were the most common serotypes in sewage, whilst CBV-5, EV-11, coxsackievirus A9, EV-22, CBV-3 and EV-30 were the most common clinical isolates. Reasons for the differences are not known but several explanations are possible. Seasonal variation of enterovirus occurrence in both sources showed an expected peak in the autumn with a trough in the spring. The occurrence of enteroviruses was closely correlated with monthly recordings of mean relative humidity. A further observation concerning the clinical specimens in Finland was the relative excess of some serotypes, such as echovirus 22 and coxsackievirus A9, and paucity of others, for instance, echoviruses 4 and 9, when compared to published data from other countries. This is consistent with the idea of geographically restricted circulation of enteroviruses.

Genetic divergence of poliovirus strains isolated in the Karachi region of Pakistan.

Seventy-seven wild poliovirus strains isolated from poliomyelitis cases in the Civil Hospital of Karachi in Pakistan in 1989-1993 were selected for partial sequence analysis covering the VP1/2A junction region of the viral genome to study the genetic relationships and epidemiological links between strains. Viral RNA was partially amplified by RT-PCR and sequenced by a solid phase method. Computer analysis revealed genetic divergence of the strains within each serotype. Most of the nucleotide differences between strains were silent: only a few specific amino acid substitutions were seen in the sequenced region. Three genotypes of poliovirus type 1 and two of poliovirus type 3 were co-circulating, while type 2 strains were represented by a single genotype. Representatives of all the genotypes present have been found among previously or concurrently characterized stains isolated elsewhere, but direct epidemiological links were found only in the case of serotype 1. Many of the epidemics caused by poliovirus type 1 in other countries were genetically linked to Pakistan. This study clearly shows the endemic circulation and wide variation of all three poliovirus serotypes in southern Pakistan and indicates the need for more effective vaccination programmes to prevent the further spread of these wild viruses.

Topical treatment of recurrent mucocutaneous herpes with ascorbic acid-containing solution.

We carried out a randomized double-bind, placebo-controlled clinical trial on the topical treatment of recurrent mucocutaneous herpes with a strong water solution of Ascoxal, an ascorbic acid-containing pharmaceutical formulation with mucolytic and non-specific antimicrobial activities. The lesion was firmly pressed with a cotton wool pad soaked in drug solution 3 times for 2 min with 30-min intervals on the first day only. Evaluation of the effects was by daily recordings of several different symptoms, including the presence and severity of erythema, induration, papulae or vesicles and scab by both the patient and a trained nurse, and by virus culture. Fourteen episodes with active treatment and 18 with the placebo were analyzed. According to the patients' records, the active treatment resulted in a significantly smaller cumulative number of days with scab (P < 0.01), or with any remaining symptom (P < 0.02) and significantly fewer occasions of worsening of any symptom after the treatment (P < 0.05). According to the nurse's records, the persistence of scabs was significantly shorter in the active treatment group (means 3.4 vs 5.9 days, P = 0.03). Virus culture after the first day of treatment yielded herpes simplex virus significantly less frequently in the active treatment group than in the placebo group (P < 0.01). In conclusion, a brief treatment with this ascorbic acid-containing preparation resulted in statistically significant clinical and antiviral effects, which calls for further and more extensive studies with a more intensive treatment schedule.

Selective isolation of poliovirus in recombinant murine cell line expressing the human poliovirus receptor gene.

Sixty-eight laboratory strains representing 49 enterovirus, 10 adenovirus, and 3 reovirus serotypes were inoculated in a recombinant murine cell line expressing the human poliovirus receptor gene (L alpha cells). Only polioviruses caused cytopathic effect over a 10-day period. Likewise, only polioviruses were isolated, by use of L alpha cells, from 168 fecal specimens from children from developing countries. These results suggest that the recombinant L alpha cells can be used for selective isolation of poliovirus from clinical specimens.

Intrathecal immune response and virus-specific immunoglobulin M antibodies in laboratory diagnosis of acute poliomyelitis.

The intrathecal immune response in 114 patients with clinically diagnosed acute poliomyelitis was studied by measuring poliovirus-specific immunoglobulin M (IgM) antibodies in cerebrospinal fluid (CSF) by a mu-capture immunoassay and by assessing the ratio between levels of poliovirus-neutralizing antibodies in serum and CSF. Fecal specimens were used for attempts to isolate the causative agents. Eighty-five percent of CSF specimens collected during the first 15 days of disease contained virus-specific IgM antibodies. Forty-five of 48 tested children (94%) also showed virus-specific IgM responses in their sera. Later on, the antibody levels decreased, and positive results after 30 days of onset of paralytic symptoms were rare. If the presence of poliovirus-specific IgM antibodies in the CSF was considered diagnostic, more cases were confirmed by this test than by virus isolation. A relative increase in poliovirus-neutralizing antibodies in the CSF was observed in about one-third of the cases; in all but three cases the increase was observed together with the presence of virus-specific IgM antibodies. A systemic virus-specific response can be seen and poliovirus can be isolated from a subclinically infected individual suffering from a concomitant poliomyelitis-like disease, while positive results by the two methods demonstrating an intrathecal immune response are likely to indicate a true causal relationship between infection and disease. Demonstration of poliovirus-specific IgM antibodies in the CSF thus appears to be a sensitive and specific method for laboratory confirmation of clinically diagnosed poliomyelitis.

Seasonal variation in etiology of travelers' diarrhea. Finnish-Moroccan Study Group.

The etiology of travelers' diarrhea was studied in 579 adult Finnish tourists participating in two packaged tours to Morocco in the winter (n = 233) and fall (n = 346) of 1989. A research team accompanied the travelers, and a laboratory for enteric pathogens was established in Agadir. At least one pathogen was found in 62% of the 60 diarrhea cases in winter and in 58% of the 111 diarrhea cases in fall. Multiple pathogens were found less often in winter (8%) than in fall (21%, P less than .05). Campylobacter strains were the leading cause of travelers' diarrhea in winter, found alone or with other pathogens in 28% of the cases (but in only 7% in fall), whereas enterotoxigenic Escherichia coli (ETEC) was the most common pathogen in fall, present in 32% of the cases (8% in winter). Both differences are highly significant (P less than .001). Salmonella enterica was almost as common as ETEC in fall (25% of diarrhea cases) but rare in winter (10%, P less than .05). Thus, the etiology of travelers' diarrhea varied according to the season in the same tourist destination. This finding has relevance to both antimicrobial treatment and prophylaxis.

Poliovaccine virus in the cerebrospinal fluid after oral polio vaccination.

In February-March 1985 an oral poliovirus vaccine campaign was launched in Finland in a population vaccinated earlier with inactivated poliovaccine. During this campaign a strain of poliovirus was isolated from the cerebrospinal fluid (CSF) of a 7-year-old girl 34 days after she had received oral poliovirus vaccine. She had long-lasting headache, vomiting and fever but no paralysis. This case demonstrates that poliovaccine virus can invade the central nervous system even after a complete course of inactivated poliovirus vaccine if the inactivated vaccine has been poorly antigenic against one of the three types of virus.

Simultaneous administration of Haemophilus influenzae type b capsular polysaccharide-diphtheria toxoid conjugate vaccine with routine diphtheria-tetanus-pertussis and inactivated poliovirus vaccinations of childhood.

A Haemophilus influenzae type b capsular polysaccharide-diphtheria toxoid conjugate vaccine (PRP-D) is capable of protecting infants against invasive H. influenzae diseases. Therefore it is very likely that it will be incorporated in routine vaccination schedules during the next few years. In order to test the suitability of simultaneous administration of PRP-D and other vaccines we administered it to 25 infants mixed with diphtheria-tetanus-pertussis vaccine at 3, 4 and 6 months and simultaneously, but in a separate syringe, with inactivated polio vaccine at 12 months. A comparison group of equal size received only diphtheria-tetanus-pertussis and inactivated poliovirus vaccines. The concentration of postvaccination antibodies to diphtheria toxoid was 0.411 IU/ml in the group that received PRP-D vs. 0.352 IU/ml in the comparison group, to tetanus toxoid 3.666 vs. 3.668 IU/ml and the neutralization titer to poliovirus type 1 was 370 vs. 320 units in the comparison group, to type 2 titer values were 230 vs. 270 units and to type 3, respectively, 210 vs. 290 units. Thus the seroresponse to antigens in routine vaccines was not affected by the presence of PRP-D in the vaccination schedule, and PRP-D can safely and effectively be included in the vaccination schedule of infancy.