Production-level risk factors for syncytial hepatitis in farmed tilapia (Oreochromis niloticus L).

Syncytial hepatitis (SHT) is an emerging viral disease of tilapia characterized by significant morbidity and mortality. This study aimed to establish the production-level risk factors associated with presence and severity of SHT. Production factors were analysed during multiple outbreaks of SHT that occurred between 2011 and 2013 on a single tilapia farm in Ecuador and compared with the year 2010 before the SHT outbreaks. Relative risks, t tests, modified Poisson and forward stepwise linear regression analyses were performed using EPIINFO™. Compared to other strains, Chitralada had an elevated risk of SHT [RR = 2.1 (95%CI 1.8-2.4)]. Excessive mortality associated with the presence (and severity) of SHT increased by 611 (365), 6,814 (5,768) and 388 (340) deaths per 100,000 fry when stocking density, dissolved oxygen and pond production cycles were raised by 1 fish/m2 , 1 mg/L and 1 cycle, respectively. Excessive mortality associated with the presence (and severity) of SHT decreased by 337 (258) and 1,354 (1,025) deaths per 100,000 when stocking weight and water temperature increased by 1 g and 1°C, respectively. Time (season and stocking year) was not significantly associated with SHT. This study shows that some production factors increase the risk incidence and severity of SHTon a farm.

Existence of Angiostrongylus cantonensis in rats (Rattus norvegicus) in Grenada, West Indies.

The zoonotic rat lung worm, Angiostrongylus cantonensis is endemic to Asia, North America, Africa and Australia. The parasite is expanding geographically and has recently been recorded in some of the Greater Antilles in the northern part of the Caribbean. In this study A. cantonensis is reported for the first time in the Lesser Antilles in one of the southernmost islands, Grenada. Between September 2005 and September 2006, 192 rats (Rattus norvegicus) were trapped throughout the island. The rats were anesthetized, exsanguinated, necropsied and the lungs were fixed whole in 10% buffered formalin, trimmed, processed, cut at 3microm, stained with hematoxylin and eosin and examined microscopically. A total of 45 (23.4%) of the 192 rats examined were found to be infected with A. cantonensis and adult worms were found in the cardiopulmonary system of one of the rats. Microscopically, pulmonic lesions, consisting of pulmonary thrombosis, hypertrophy of pulmonary arteries and granulomatous pneumonia were associated with intralesional adults, larvae and embryonated eggs of A. cantonensis. An incidental finding of variably sized (2-7mm) solitary to multiple cysts containing larvae of Taenia taeniaformis were seen in the livers of 57 rats. This report of A. cantonensis in Grenada provides evidence of the further global expansion of this important zoonotic parasite and the public health implications of this discovery is discussed.

Progress towards international adoption of the World Health Organization ultrasound classification of cystic echinococcosis.

Cystic echinococcosis (CE) is a global parasitic zoonosis for which ultrasound (US) is the gold standard modality for diagnosis. In 2003, the WHO published a standardized US classification of CE, on which WHO treatment guidelines are based. In 2014, global adoption of the classification was questioned by a publication which indicated that, between 2004 and 2014, only half of studies utilizing a classification used the WHO classification. More recent studies have demonstrated that the WHO classification best reflects the natural history of CE, and is used with high reliability by experts in the field; despite these attributes, the classification's impact is ultimately limited by the extent of its adoption. A PubMed search using the terms "Echinococcus granulosus ultrasound," "Echinococcus granulosus classification," "cystic echinococcosis ultrasound," and "cystic echinococcosus classification" revealed publications on human CE utilizing a US classification. Classification(s) used, year of publication, and the country of the first author's institution were recorded. From 2004 to 2010, the WHO classification was used in 50% or fewer of included publications for 6 of the 7 years. After 2011, it appeared in a low of 75% (2013) to a high of 96% (2017) of included publications. Of all included studies published from 2004 to 2017, the WHO classification was referenced in 18% (3 of 17) from Africa, 64% (32 of 50) from Asia, 79% (89 of 113) from Europe, 89% (8 of 9) from North America, and 100% (9 of 9) from South America. Findings suggest that the WHO classification has been progressively taking preference to other classifications, with rate of adoption depending on continent of origin of the research. Residual use of the classification developed by Dr. Hassen Gharbi of Tunisia in 1982, used widely prior to development of the WHO classification (which reversed two stages in Gharbi's classification in order to more closely reflect the natural history of CE) suggests that adoption of a new classification takes time and varies regionally.

Cystic echinococcosis in Turkana, Kenya: 30 years of imaging in an endemic region.

Cystic echinococcosis (CE), a widespread, complex zoonosis, causes chronic disease associated with high morbidity. The pastoral Turkana people of Kenya have one of the highest prevalence rates of CE in the world. Between 1983 and 2015, a CE control program in the Turkana region used ultrasound (US) screening surveys and surgical outreach visits to evaluate CE prevalence and treat those with the disease. As the gold standard modality for diagnosing CE, US reveals a great deal of information about the disease in affected populations. The aim of this study is to discuss the characteristics of untreated CE in the Turkana people as revealed by US data collected during the CE control program and evaluate disease presentation, factors influencing the risk of transmission, and the timeline of disease progression. Data were obtained from written patient notes from US screenings and images; cysts were classified using the World Health Organization (WHO) standardized US classification of CE. Findings include greater prevalence of cysts, later stages of cysts, and multiple cysts in older age groups, with no multiple cysts occurring in patients under six years of age, which are consistent with the assertion that rates of exposure, transmission, and infection increase with age in endemic regions. Findings also raise questions regarding the timeline of disease progression, and factors potentially influencing disease transmission within this and other endemic populations. A comprehensive survey focusing on cultural and community observations (e.g., changing behaviors, hygienic practices, etc.) may provide more detailed information regarding factors that facilitate transmission.

The natural history of cystic echinococcosis in untreated and albendazole-treated patients.

The World Health Organization (WHO) treatment protocols for cystic echinococcosis (CE) are based on the standardized ultrasound (US) classification. This study examined whether the classification reflected the natural history of CE in untreated and albendazole-treated patients. Data were collected during mass US screenings in CE endemic regions among transhumant populations, the Turkana and Berber peoples of Kenya and Morocco. Cysts were classified using the WHO classification. Patient records occurring prior to treatment, and after albendazole administration, were selected. 852 paired before/after observations of 360 cysts from 257 patients were analyzed. A McNemar-Bowker χ2 test for symmetry was significant (p<0.0001). 744 observations (87.3%) maintained the same class, and 101 (11.9%) progressed, consistent with the classification. Regression to CE3B occurred in seven of 116 CE4 cyst observations (6.0%). A McNemar-Bowker χ2 test of 1414 paired before/after observations of 288 cysts from 157 albendazole-treated patients was significant (p<0.0001). 1236 observations (87.4%) maintained the same class, and 149 (10.5%) progressed, consistent with the classification. Regression to CE3B occurred in 29 of 206 CE4 observations (14.1%). Significant asymmetry confirms the WHO classification's applicability to the natural history of CE and albendazole-induced changes. Regressions may reflect the stability of CE3B cysts.

Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies.

Cats are important in the natural epidemiology of Toxoplasma gondii, because they are the only hosts that can excrete environmentally resistant oocysts. Cats are infected with T. gondii via predation on infected birds and rodents. During 2005, 238 rats (Rattus norvegicus) were trapped in Grenada, West Indies, and their sera along with tissue samples from their hearts and brains were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test (MAT, titer 1:40 or higher); only 2 (0.8%) of 238 rats were found to be infected. Brains and hearts of all rats were bioassayed in mice. Toxoplasma gondii was isolated from the brain and the heart of only 1 rat, which had a MAT titer of 1:320. All of 5 mice inoculated with the heart tissue, and the 5 mice inoculated with the brain tissue of the infected rat remained asymptomatic, even though tissue cysts were found in their brains. Genetically, the isolates of T. gondii from the heart and the brain were identical and had genotype III by using the SAG1, SAG2, SAG3, BTUB, and GRA6 gene markers. These data indicate that rats are not important in the natural history of T. gondii in Grenada.

Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from chickens in Grenada, West Indies.

The prevalence of Toxoplasma gondii in free-range chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 102 free-range chickens (Gallus domesticus) from Grenada was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 53 (52%) chickens with titers of 1:5 in 6, 1:10 in 4, 1:20 in 4, 1:40 in 4, 1:80 in 15, 1:160 in 9, 1: 320 in 5, 1:640 in 4, and 1:1,280 or greater in 2. Hearts, pectoral muscles, and brains of 43 seropositive chickens with MAT titers of 1:20 or greater were bioassayed individually in mice. Tissues of each of 10 chickens with titers of 1:5 and 1:10 were pooled and bioassayed in mice. Tissues from the remaining 49 seronegative chickens were pooled and fed to 4 T. gondii-free cats. Feces of cats were examined for oocysts; they did not shed oocysts. T. gondii was isolated from 35 of 43 chickens with MAT titers of 1:20 or greater; from the hearts, brains, and pectoral muscles of 2, hearts and brains of 20, from the hearts alone of 11, and brains alone of 2. T. gondii was isolated from 1 of 10 chickens with titers of 1:5 or 1:10. All 36 T. gondii isolates were avirulent for mice. Genotyping of these 36 isolates using polymorphisms at the SAG2 locus indicated that 29 were Type III, 5 were Type I, 1 was Type II, and 1 had both Type I and Type III. Genetically, the isolates from Grenada were different from those from the United States; Type II was the predominant type from the United States. Phenotypically, all isolates from Grenada were avirulent for mice, whereas those from Brazil were mouse-virulent. This is the first report of isolation of T. gondii from chickens from Grenada, West Indies.

Beta-haemolytic streptococci in school children 5-15 years of age with an emphasis on rheumatic fever, in the tri-island state of Grenada.

The objective of this study is to determine the prevalence of beta-haemolytic streptococci in the pharynx and antibodies to beta-haemolytic streptococci in school children 5-15 years of age in the tri-island state of Grenada Blood samples and throat swabs were obtained from 1388 school children, aged 5-15 years old attending randomly selected schools in each parish of the tri-island state of Grenada. Serum samples were subjected to antistreptolysin o testing (ASOT) while throat swabs were cultured on sheep blood agar plates. The prevalence of positive throat swab was 15.4% (95% CI = 13.4%, 17.2%). The rate was highest in St Patrick (21.8%) and lowest in Carriacou (5.7%). The prevalence of antibodies was 38.6% (95% CI = 37.6%, 42.8%). It was highest in St Patrick (54.4%) and lowest in Petit Martinique (26.8%). In St Patrick, males were significantly more likely than females to have a positive ASOT (p = 0.0084). In St George's, males were significantly more likely than females to have a positive throat culture (p = 0.0172). Thirty-four per cent of the positive cultures were type A, 10% were type C and 56% type G. The data illustrate that there is a high prevalence of beta-haemolytic streptococci in school children in certain parishes in Grenada. Public health measures should address prevention and control of beta3-haemolytic streptococcal infection in order to prevent the possible sequelae of this disease.

An active intermediate host role for man in the life cycle of Echinococcus granulosus in Turkana, Kenya.

Hydatid material removed at operation from 97 Turkana was compared for fertility and viability with hydatid material removed from camels, cattle, sheep, and goats. The results showed that the human material was extremely fertile and viable, as was material from the camels, goats, and sheep. Cattle cysts were invariably sterile, and the protoscoleces, when present, were comparatively less viable. The high incidence and fertility of hydatid cysts in the Turkana, together with the lack of burial customs thus allowing dogs to have ready access to infected human corpses, means that the Turkana are potential biological participants in the cyclic transmission of Echinococcus granulosus in this region. This is a unique situation, for elsewhere in the world man is regarded as an accidental host who plays no role in the parasite's life cycle.

The identification of eggs of Echinococcus by immunofluorescence using a specific anti-oncospheral monoclonal antibody.

A relatively simple and specific test has been developed to distinguish eggs of Echinococcus from those of other morphologically identical taeniid species. A specific anti-Echinococcus oncosphere monoclonal antibody was produced which binds in an indirect immunofluorescence test to egg-derived oncospheres of E. granulosus but not to those of other taeniid species, such as Taenia hydatigena, T. saginata, T. pisiformis, T. ovis, T. multiceps, or T. taeniaeformis. Specific fluorescence was obtained with oncospheres of E. granulosus derived from either hatch/activated viable eggs using artificial intestinal fluid or from hypochlorite/detergent treated eggs. The potential use of this test in the study of the transmission of Echinococcus in Turkana, Kenya, is discussed.

An extensive ultrasound and serologic study to investigate the prevalence of human cystic echinococcosis in northern Libya.

A prevalence study of abdominal cystic echinococcosis (CE) was undertaken in the northwest, north-central, and northeast regions of Libya. A total of 36 villages along the coast were included, in which 20,220 people were screened by portable ultrasound. Three hundred thirty-nine (1.7%) were diagnosed with CE. There was no significant difference between CE prevalence rates in the three regions of Libya (mean = 1.6%); however, intervillage rates were variable, ranging from 0% to 4.5%. The prevalence of CE increased significantly with age (P < 0.0001) and females were significantly more affected (2%) than males (1.3%) (P < 0.0001). Cases of CE were distributed among 3.2% of housewives, 2.6% of farmers, 2.3% of male civil servants, 1.3% of female students, and 1.1% of male students. Housewives (P < 0.0001) and students (P < 0.0001) were significantly more at risk for CE. A statistically significant proportion (62% [210 of 339]) of CE cases kept dogs (P < 0.0001). Of the ultrasound-positive CE cases 69% (233 of 339) were antibody seropositive by ELISA using Echinococcus granulosus hydatid cyst fluid antigen B. Blood samples (n = 10,096) collected onto filter papers were taken randomly from ultrasound-negative people who entered the study, of which 11.2% were found to be seropositive. Seropositivity increased with age (P < 0.0001), with females exhibiting higher seroprevalence than males for all age groups (P < 0.0001). Seroprevalence was distributed occupationally among 17.3% of female students, 11.2% of male students, 8.3% of housewives, 7.3% of farmers, and 6.4% of male civil servants. Housewives and students were more likely to be seropositive (P < 0.0001). Forty-seven percent (526) of the seroreactors kept dogs. All liver hydatid cysts detected by ultrasound during community screening were classified according to morphology and size into six types. Type I (17.5% of all cases) were small univesicular cysts less than 50 mm in diameter with no laminations or daughter cysts. Type II (34%) were univesicular cysts with only laminations. Type IIIa (8.5%) were univesicular cysts with the appearance of laminations and daughter cysts. Type IIIb (13%) were univesicular with laminations and less prominent daughter cysts. Type IV (5.6%) presented as a solid mass. Type V were degenerated calcified or partially calcified cysts (13.2%). Type VI presented as multiple cysts (8% of all cases). The CE cases that exhibited Types II, IIIa, IIIb, IV or VI cysts showed the highest seropositivity (86%, 96%, 95%, 100%, and 96%, respectively), while Types I and V were the least seroreactive (38% and 22%, respectively). Cases of CE occurred in 311 families, with 93% having only one member as a CE case while 7% of the families had two or more cases. However, 25% of the ultrasound-negative persons belonging to families with an index CE case were seropositive for antibodies to Echinococcus. These results confirm the importance of human CE in Libya. They also confirm the usefulness of ultrasound combined with serology as a mass screening approach for CE in north African communities.

The existence of Dracunculus medinensis (Linnaeus, 1758) in Turkana, Kenya.

Dracontiasis has been previously reported in southern Sudan, northern Uganda and north-west Eritrea but this is the first report of autochthonous cases in Turkana, Kenya. Five Turkana tribesmen, three females from the same family, one young boy and an adult male were being treated for guinea-worm at the dispensary in Lokichogio, northern Turkana District. The three women had recently returned from southern Sudan, where the disease is endemic, but the two male tribesmen had only lived in and around the Lokichogio region. The methods used in obtaining water from water-holes dug in dry river beds provide an ideal situation for Dracunculus transmission amongst the tribesmen; this parasite may therefore become a problem in this remote impoverished area.

Epidemiology of hydatid disease in Kenya: a study of the domestic intermediate hosts in Masailand.

The results are given of a study on the epidemiological significance of cattle, sheep and goats in maintaining the life-cycle of Echinococcus granulosus in Masailand, Kenya. A total of 134 (8.9%) of 1499 cattle, 146 (8.1%) of 1798 sheep and 144 (7.1%) of 2020 goats slaughtered in abattoirs in Masailand were found to harbour hydatid cysts. The frequency distribution of the hydatid cysts in each intermediate host species was over-dispersed. Of 729 cattle cysts examined 71 (9.7%) were fertile (cyst contained protoscoleces) with an average viability of 71%, 507 (69.5%) were sterile and 151 (20.7%) were calcified. Out of 810 sheep and 451 goat cysts examined 571 (70.5%) and 213 (47.0%) were fertile with respective viabilities of 88% and 86% and 143 (17.7%) and 194 (43.0%) were sterile leaving 96 (11.6%) and 45 (10.0%) sheep and goat cysts which were calcified. The greater fertility of the sheep and, to a lesser extent, goat cysts coupled with the reported greater infectivity of sheep and goat than cattle material to dogs and the common Masai practice of slaughtering sheep and goats at home, indicates that, at present, sheep and goats are the most important intermediate hosts for the maintenance of the domestic life-cycle of E. granulosus in Masailand. The incidence of hydatidosis in donkeys and the relationship of the domestic cycle to the wildlife cycle operating in the same area is unknown and requires further study.

Pastoralists and hydatid disease: an ultrasound scanning prevalence survey in east Africa.

An attempt was made to estimate the prevalence of hydatid disease in nomadic pastoralists living in eastern Africa and to identify environmental, cultural and behavioural factors which may influence Echinococcus transmission. 18,565 nomadic pastoralists, from 12 different groups living in the vast, semi-desert regions of Kenya, Sudan, Ethiopia and Tanzania, were screened for hydatid cysts using a portable ultrasound scanner. High prevalences of hydatidosis were recorded among the north-western (5.6%) and north-eastern (2.1%) Turkana of north-west Kenya, the Toposa (3.2%) of southern Sudan, the Nyangatom (2.2%), Hamar (0.5%) and Boran (1.8%) of south-west Ethiopia and northern Kenya and the Maasai (1.0%) of Tanzania. Lower prevalences were recorded amongst the southern (0.3%) and lake dwelling (0.3%) Turkana and the Pokot (0.1%) of Kenya. The disease was not found amongst the Turkana, Samburu, Dassanetch, Gabbra, Somali or Rendille screened on the east side of Lake Turkana. The scanning surveys were well accepted by the people and provided evidence for the need to expand the present hydatid control programme in Turkana to cover the whole hyperendemic focus. Such a programme must contain an educational component for, although most groups recognized hydatid cysts, there was complete lack of knowledge concerning the parasite and its mode of transmission.

Immunodetection of Echinococcus eggs from naturally infected dogs and from environmental contamination sites in settlements in Turkana, Kenya.

A species-specific indirect immunofluorescence test using an anti-Echinococcus oncosphere monoclonal antibody (EgOH6-4E5) was applied to identify Echinococcus oncospheres released from taeniid eggs collected in environmental soil and water samples, and from perianal or faecal samples of naturally infected dogs, in northern Turkana, Kenya. The specificity of immunodetection of Echinococcus eggs by specific fluorescence of Echinococcus oncospheres from naturally infected dogs was 100% when compared to Taenia hydatigena infections, and a sensitivity of 73% was obtained in the detection of dogs infected with Echinococcus using perianal Scotch tape swabs. Taeniid eggs were recovered from various soil samples inside Turkana manyattas (settlements) and from waterhole samples. Some oncospheres obtained from taeniid eggs recovered from all sampled areas, but particularly from inside Turkana huts (akai) and from water samples from open waterholes used by the people and their livestock, reacted positively with the Echinococcus 4E5 monoclonal antibody. The potential importance of contamination of such sites with Echinococcus eggs is discussed in relation to the transmission of echinococcosis in this hyperendemic region of northern Kenya.

Hydatid disease: research and control in Turkana, IV. The pilot control programme.

A pilot programme to control hydatid disease in north-west Turkana district was started in October 1983 with an intensive educational campaign. This was followed by control and treatment of the dog population to reduce the prevalence of Echinococcus granulosus, thereby reducing the infective pressure on man. Surveillance is by mass annual sero-epidemiological and ultrascan surveys of the nomadic Turkana within the control region and by arecoline purging of the dogs. Some of the recent advances in research on hydatid disease in subject areas such as immunodiagnosis, chemotherapy and epidemiology have been incorporated into the present programme, and their relevance to this and other similar programmes is discussed.

Ultrasound scanning for detecting morbidity due to Schistosoma haematobium and its resolution following treatment with different doses of praziquantel.

A study to assess the resolution of urinary tract morbidity due to Schistosoma haematobium was conducted on 2 cohorts of schoolchildren attending neighbouring schools in Kilombero District, southern Tanzania. Schoolchildren were screened for S. haematobium infection using the standard World Health Organization filtration technique and subsequently examined for urinary tract pathology using a portable 3.0 MHz sector scanner (Siemens Sonoline 1300). Treatment with praziquantel was given to all infected children. Children with observed urinary tract pathology received either 20 (n = 52) or 40 (n = 79) mg/kg body weight and were sonographically re-examined one, 2, 3 and 6 months following treatment. Geometric mean outputs of 21 and 19 eggs/ml of urine were detected in the 2 cohorts before treatment. Urinary tract pathology correlated positively with egg output (chi 2, P = 0.02) and microhaematuria (P = 0.0001). Bladder (wall irregularities and polyps) and kidney (congestive changes) pathologies were found in 81% and 36%, respectively, of the group that received 20 mg/kg of praziquantel, and in 78% and 46% of the group that received 40 mg/kg. Six months after treatment, 90.4% and 88.0% parasitological cure rates were obtained using 20 or 40 mg praziquantel/kg body weight. The respective pathology clearances were 88% and 91%. 20 mg/kg of praziquantel was as effective with regard to cure rates and reversibility of morbidity as 40 mg/kg.

In vitro resistance patterns of Plasmodium falciparum to chloroquine--a reflection of strain-specific immunity?

Studies in vitro among children on the response of Plasmodium falciparum to chloroquine were conducted as part of the national long-term monitoring of drug resistance in a holo- to hyperendemic malarious area of Tanzania between 1983 and 1989. Overall, no significant increase in chloroquine resistance was observed. However, in children under 5 years old resistance increased during this period, whereas in schoolchildren resistance decreased from 1986 to 1989. A hypothesis based on antigenic differences between resistant and sensitive strains is proposed to explain this age-specific pattern. If immunity develops principally against the most frequent parasite strains, then as it develops the numbers of the most frequent strains will be reduced, whilst, the rare strains may become predominant and thus be detected in the blood of immune patients. Thus, in an endemic area, the observed resistance pattern in non-immune infants will differ from that in immune schoolchildren, as was observed in the present study. These findings may have important implications for the control of malaria and the development of vaccines.

Cystic echinococcosis in semi-nomadic pastoral communities in north-west China.

In order to determine the prevalence of human cystic echinococcosis (CE) in semi-nomadic traditional pastoralist groups in north-west China, 2 large community studies were undertaken in Altai and Tacheng Prefectures in 1990/91 and 1995/96, respectively. The Kekergash community (Altai) comprised mainly ethnic Kazakhs, whereas the Narenhebuke community (Tacheng) comprised mainly Mongolians. Populations were screened for CE by abdominal ultrasound scan (US) and serological tests. The total prevalence of confirmed human CE was higher in Narenhebuke (2.7%, 49/1844) than in Kekergash (0.9%, 17/1861; P < 0.01). Within each community there was no significant difference of CE prevalence between the Kazakh and Mongolian groups, although Han Chinese exhibited twice the rate of CE (4.9%) in Narenhebuke compared to the dominant Mongolian population. For each community, human CE prevalence increased with age and there was a greater risk associated with the practice of home slaughter of livestock. Dogs were screened for Echinococcus granulosus infection and re-infection levels using a highly specific coproantigen test. The proportion of dogs with positive coproantigen tests was significantly higher in Narenhebuke (36.0%, 50/139) compared to Kekergash (17.8%, 16/90). In Narenhebuke the re-infection levels of dogs, as determined by coproantigen positivity, were higher in the winter quarters (49.4%, 39/79) compared to the summer quarters (18.3%, 11/60; P < 0.01). Furthermore, coproantigen re-test positivity was 25% at 3 months and 29.2% at 7 months. Highest dog coproantigen positivity was obtained over the winter period.