Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays.

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.

Prevalence of Legionella spp. in swimming pool environment.

A study was carried out to evaluate the prevalence of bacteria of the Legionella genus in the water from the pools and showers of 12 swimming pools in the city of Bologna (Italy). Sampling took place four times for every establishment, once for each season of the year. Legionella spp. were found in 2 of the 48 pool water samples (L. micdadei and L. bozenanii) and in 27 of the 48 samples taken from the hot water of the showers: 19 were positive for L. pneumophila (10-19,250 cfu/l) and 18 for other species (20-6000 cfu/l). The contamination was seen to be consistent throughout the year, since the same species of legionellae were isolated at different samplings and in concentrations that did not reveal any seasonal trend. The legionellae and the Gram negative bacteria were not found in shower water with a temperature above 43 degrees C. However, in the samples of shower water with a lower temperature a statistically significant inverse correlation was seen between legionellae and Pseudomonas aeruginosa (r = -0.51; p<0.01) as well as between legionellae and Gram negative bacteria (r = -0.70; p<0.01). The potential risk of contracting infections from Legionella spp. in the swimming pool environment does not seem to be linked to the pool water, but to that of the showers. The water temperature of showers should therefore be maintained at a level high enough to prevent the reproduction of these bacteria.

[Secondary serologic responses to dengue epidemic in 1998 in Salta, Argentina, where other flavivirus co-circulate]. / Respuestas serológicas secundarias en la epidemia de dengue de 1998 en Salta, Argentina, donde co-circulan otros flavivirus.

In 1998, a dengue outbreak (serotype 2) occurred in Salta province in Northern Argentina, following the first detection of dengue in the same area in 1997. We classified the serologic response of cases from 1998 as primary or secondary, since the risk of severe disease is greater for secondary cases. We studied 154 cases by plaque reduction neutralization and hemagglutination inhibition tests. Thirty-eight cases (25%) were classified as primary serologic responses and 84 cases (54%) as secondary responses. Thirty-two cases (21%) with borderline IgG titers could not be classified. Previous exposure to potentially cross-reacting flaviviruses (Saint Louis Encephalitis [SLE] and Yellow Fever [YF] viruses) was analyzed, as a possible cause of the secondary response pattern. Our results indicated that among cases classified as dengue secondary response, 83% could be attributed to previous SLE or YF exposure or serologic cross-reactivity. Vaccination against YF virus was at most a minor contributor to the secondary response pattern. The finding of a positive YF serologic result among persons not vaccinated may indicate silent circulation of YF in a region that can support both urban and jungle cycles. Other cases showing dengue secondary responses remained unexplained, suggesting the unrecognized occurrence of a previous infection with other dengue serotypes or of flaviviruses other than SLE or YF.

[Isolation of lymphocytic choriomeningitis virus from human individuals]. / Aislamiento del virus de la coriomeningitis linfocitaria de seres humanos.

The activity of lymphocytic choriomeningitis virus (LCMv) in Argentina has been previously reported on the basis of serological evidence in rodents and humans and the isolation of only one strain of LCMv from a Mus domesticus captured in the province of Córdoba. The aim of this paper was to register patients with serological diagnosis of LCM, to isolate and to identify human strains of LCMv in Argentina. During the last 19 years, 15 cases were diagnosed as LCM by immunoflourescent indirect assay (IFI) and enzyme-linked immunosorbent assay (ELISA) but when neutralizing assay (NT) was incorporated, eight cases were classified as confirmed, three as probable and four as negative. The geographic distribution of the cases included three provinces: Córdoba, Buenos Aires and Santa Fe. Viral isolation was attempted in five patients classified as confirmed and only two resulted positive (P5226 and P8573). They were identified as LCMv by IFI and NT. The coexistence of LCMv with other arenaviruses, such as Junin and Oliveros viruses, in the same area, raises the probability of interactions between them, which could modify the virulence and/or pathogenicity for humans associated to genomic changes. Future studies of antigenic, genomic and virulence variability of different Argentine strains of LCMv, as well as the systematic search for human infection, will contribute to define the importance of this viral agent in our country and to implement control measures.

Genetic diversity of the Junin virus in Argentina: geographic and temporal patterns.

RNA was purified from 39 strains of cell-cultured Junin virus (JUN) from central Argentina, which included both human- and rodent-derived isolates (a total of 26 and 13, respectively), as well as from 2 laboratory JUN strains, XJ Cl3 and XJ #44. JUN-specific primers were used to amplify a 511-nucleotide (nt) fragment of the nucleocapsid protein gene and a 495-nt fragment of the glycoprotein 1 (GP1) gene. Genetic diversity among JUN strains studied was up to 13% at the nt level and up to 9% at the amino acid (aa) level for the GP1 gene and up to 9% (nt) and 4% (aa) for the NP gene. Phylogenetic analyses of both genes revealed three distinct clades. The first clade was composed of the JUN strains from the center of the endemic area and included the majority of JUN strains analyzed in the current study. The second clade contained 4 JUN strains isolated between 1963 and 1971 from Cordoba Province, the western-most edge of the known endemic area. The third clade contained 4 JUN strains that originated from Calomys musculinus trapped in Zarate, the northeastern edge of the known endemic area. Certain JUN sequences, which were obtained from GenBank and identified as XJ, XJ #44, and Candid #1 strains, appeared to form a separate clade. Over 400 nt of the GP1 and GP2 genes were additionally sequenced for 7 JUN strains derived from patients with different clinical presentations and outcomes of Argentine hemorrhagic fever. Analysis of the corresponding aa sequences did not allow us to attribute any particular genetic marker to the changing severity or clinical form of the human disease.

Dengue reemergence in Argentina.

Aedes aegypti, eradicated from Argentina in 1963, has now reinfested the country as far south as Buenos Aires. In 1997, four persons with travel histories to Brazil, Ecuador, or Venezuela had confirmed dengue, and surveillance for indigenous transmission allowed the detection of 19 dengue cases in Salta Province. These cases of dengue are the first in Argentina since 1916 and represent a new southern extension of dengue virus.

Estimation of genetic divergence and gene flow between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Argentina.

Allele frequencies at seven polymorphic loci controlling the synthesis of enzymes were analyzed in six populations of Culex pipiens L. and Cx. quinquefasciatus Say. Sampling sites were situated along a north-south line of about 2,000 km in Argentina. The predominant alleles at Mdh, Idh, Gpdh and Gpi loci presented similar frequencies in all the samples. Frequencies at the Pgm locus were similar for populations pairs sharing the same geographic area. The loci Cat and Hk-1 presented significant geographic variation. The latter showed a marked latitudinal cline, with a frequency for allele b ranging from 0.99 in the northernmost point to 0.04 in the southernmost one, a pattern that may be explained by natural selection (FST = 0.46; p < 0.0001) on heat sensitive alleles. The average value of FST (0.088) and Nm (61.12) indicated a high gene flow between adjacent populations. A high correlation was found between genetic and geographic distance (r = 0.83; p < 0.001). The highest genetic identity (I(N) = 0.988) corresponded to the geographically closest samples from the central area. In one of these localities Cx. quinquefasciatus was predominant and hybrid individuals were detected, while in the other, almost all the specimens were identified as Cx. pipiens. To verify the fertility between Cx. pipiens and Cx. quinquefasciatus from the northern- and southernmost populations, experimental crosses were performed. Viable egg rafts were obtained from both reciprocal crosses. Hatching ranged from 76.5 to 100%. The hybrid progenies were fertile through two subsequent generations.

Genetic diversity and epidemiology of hantaviruses in Argentina.

Phylogenetic analysis of a 292-nucleotide (nt) fragment of the hantavirus M genome segment from 36 rodent and 13 human samples from three known foci of hantavirus infection in Argentina was conducted. A 1654-nt fragment of the M genome segment was analyzed for 1 representative of 7 genetically distinct hantavirus lineages identified. Additionally, the nt sequence of the complete M genome segments of Lechiguanas, Oran, and Hu39694 hantavirus genotypes was determined. nt sequence comparisons reveal that 7 hantavirus lineages from Argentina differ from each other by 11.5%-21.8% and from Sin Nombre, Bayou, and Black Creek Canal viruses by 23.8%-26.5%. Phylogenetic analyses demonstrate that they form a unique, separate branch within the clade containing other New World sigmodontine-borne hantaviruses. Most Oligoryzomys-borne hantavirus genotypes clearly map together. The Oligoryzomys-borne genotypes Lechiguanas, Oran, and Andes appear to be associated with human disease. Oligoryzomys longicaudatus was identified as the likely rodent reservoir for Andes virus.

Characterization of arenaviruses using a family-specific primer set for RT-PCR amplification and RFLP analysis. Its potential use for detection of uncharacterized arenaviruses.

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.

Biological characteristics of an enzootic subtype of western equine encephalomyelitis virus from Argentina.

In order to expand our knowledge on the biological characteristics of an enzootic South American subtype of western equine encephalomyelitis (WEE) virus, strain AG80-646, we inoculated guinea pigs, rabbits, newborn chickens and Vero and chick embryo cell cultures with this and other WEE and Wee-related viruses. AG80-646 was found apathogenic for guinea pigs even when inoculated intracranially (i.e.) or intraperitoneally (i.p.), and the animals did not develop viraemia. AG80-646 killed rabbits and the animals developed high viraemia (peak titer was 7.0 log PFU/0.1 ml). These data and previous serological evidence led us to look for a mammal as a natural host. AG80-646 was found lethal for newborn chickens inoculated subcutaneously (s.c.) (peak viraemia titer was 6.6 log PFU/0.1 ml). AG80-646 produced plaques (diameter 0.8-1.0 mm) in Vero and chick embryo cells 3-4 days post infection (p.i.) A comparison of AG80-646 with other WEE complex virus strains led to the following observations: (1) The lethality for guinea pigs was high for the two epizootic Argentinian strains, Cba 87 and Cba CIV 180, zero for the two enzootic strains, AG80-646 and BeAr 10315 (virus Aura), and intermediate for the Russian strain Y62-33 (low by i.c. route and zero by i.p. route); (2) AG80-646 was more virulent for rabbits inoculated i.p. than the three epizootic strains Cba 87, Cba CIV 180 and McMillan; (3) AG80-646 was less virulent for new-born chickens than the Argentinian epizootic strain Cba CIV 180; (4) The viraemia level correlated always with the strain virulence in each animal host. This study provides tools for the differentiation of WEE complex viruses and strains in the future ecological work on WEE in South America.

[Retrospective detection of hantavirus clinical infections in Argentina]. / Detección retrospectiva de infecciones clínicas por hantavirus en la Argentina.

Hantavirus activity in rodents and human beings in Argentina has been known since the 1980's. In this study, we retrospectively investigated hantavirus infections among Argentine Hemorrhagic Fever (AHF) cases notified between 1987 and 1994, without virological confirmation. IgG and IgM antibodies to hantavirus were tested by ELISA. Among 1028 patients included in the study, we found 13 recent infections (1.26%) and 13 remote infections (1.26%). IgG antibodies determined in 745 healthy persons living in the same localities of recent infection cases, gave only one positive result (0.13%). Nine of the 13 recent infections had the clinical presentation of Hemorrhagic Fever with Renal Syndrome (HFRS) while the other four were in the form of Hantavirus Pulmonary Syndrome (HPS). We performed a clinical and epidemiological comparison between the nine patients with FHSR and two paired control groups: one with confirmed AHF and the other with Febrile Syndrome of Undetermined Etiology (FSUE), which were negative for hantavirus, Junin and LCM. There were no differences between clinical signs or symptoms. Nevertheless, normal or high leucocyte counts, with thrombocytopenia, hemoconcentration, high creatinine levels and proteinuria in HFRS cases resulted useful for differential diagnosis. These results showed the coexistence of Junin virus and hantaviruses in the endemic area of AHF, and indicate the importance of including the infection with these viruses in the differential diagnosis of hemorrhagic fevers and respiratory distress syndromes of unknown etiology. The clinical variability found could be related to the presence of more than one hantavirus serotype in our country.

The role of mononuclear blood cells in experimental Junin virus spread to the central nervous system.

The neuroinvasiveness of Junin virus depends on the viral strain, animal species, and age. The role of infected blood cells in hematogenous Junin virus spread to the central nervous system (CNS) was studied by determining the growth in pheripheral mononuclear cells and brain tissue of Candid 1 and XJCL3 laboratory strains, in Calomys musculinus and guinea pigs. The present study demonstrated that Junin virus replicates in circulating peripheral lymphocytes and macrophages of 11-day-old guinea pigs and 6 +/- 1-day-old Calomys musculinus. Moreover, the observation that mononuclear phagocyte depletion did block Junin virus neuroinvasion firmly indicates that the cellular viremia (circulating monocytes) is one of the mechanisms of Junin virus attenuated strains spread to the CNS in animal hosts.

Western equine encephalomyelitis: virulence markers and their epidemiologic significance.

Comparative studies are described on the virulence of the western equine encephalomyelitis (WEE) complex viruses for mice. Three epizootic WEE virus strains (McMillan, Cba 87, and Cba CIV 180) and five enzootic WEE complex viruses (Highlands J [HJ], Y62-33, Aura, Fort Morgan [FM], and WEE AG80-646) were examined. The neurovirulence and the neuroinvasiveness of these viruses for adult mice were established and correlated with viremia and virus replication in brain tissue. Adult mice inoculated intraperitoneally showed differential responses that corresponded to the epidemiologic attributes of WEE viruses. Viruses associated with equine epizootics were neurovirulent and neuroinvasive, whereas enzootic viruses were neither neuroinvasive nor neurovirulent. In North America, HJ virus appears to be an antigenic link with an intermediate virulence between epizootic WEE virus and the enzootic FM virus. The HJ virus has been associated with rare cases of sporadic equine and human diseases. In South America, no virus with intermediate virulence characteristics has been described. We speculate that epizootics may arise from nonpathogenic strains such as AG80-646 maintained in enzootic transmission cycles.

[Post-epizootic activity of Western equine encephalitis virus in Argentina]. / Actividad post-epizoótica del virus de la encefalitis equina del oeste en la Argentina.

It is shown here the WEE virus activity in ARgentina in 1983-1986 post-epizootic period. A surveillance system was established by the equine case notification and the sentinel animal method. Among the thirteen equine focus notified between September 1983-September 1985 in Córdoba and Santa Fe Provinces, 5 presumptive cases out of 16 sick horses were confirmed by the hemagglutination inhibition test for WEE epizootic virus. Twenty eight notified human cases were studied with negative results. The neutralizing antibody (Ac NT) prevalence among sentinel horses in Córdoba Province (4%) was lower than that of Santa Fe Province (13%) in April, 1984. It was shown that Ac NT disappeared in a 12-month period in 40% of the Santa Fe horses. Two seroconversions in Córdoba (12/1983 to 4/1986) and 9 seroconversions in Santa Fe (2/1984 to 7/1986) were registered. One of the sentinel horses form Córdoba Province also seroconverted with the enzootic WEE AG 80-646 virus. Due to the recognized cross-antigenicity between both viruses, reactivity was attributed to WEE epizootic virus. No viral activity was detected in the domestic birds. With this contribution, the study of one epizootic cycle of WEE virus was completed: pre-epizootic silence (1977-1980), epizootic (1982-1983) and residual focus plus inapparent infections during a post-epizootic period (1983-1986).

Actividad post-epizoótica del virus de la encefalitis equina del oeste en la Argentina / [Post-epizootic activity of Western equine encephalitis virus in Argentina]

It is shown here the WEE virus activity in ARgentina in 1983-1986 post-epizootic period. A surveillance system was established by the equine case notification and the sentinel animal method. Among the thirteen equine focus notified between September 1983-September 1985 in Córdoba and Santa Fe Provinces, 5 presumptive cases out of 16 sick horses were confirmed by the hemagglutination inhibition test for WEE epizootic virus. Twenty eight notified human cases were studied with negative results. The neutralizing antibody (Ac NT) prevalence among sentinel horses in Córdoba Province (4

) was lower than that of Santa Fe Province (13

) in April, 1984. It was shown that Ac NT disappeared in a 12-month period in 40

of the Santa Fe horses. Two seroconversions in Córdoba (12/1983 to 4/1986) and 9 seroconversions in Santa Fe (2/1984 to 7/1986) were registered. One of the sentinel horses form Córdoba Province also seroconverted with the enzootic WEE AG 80-646 virus. Due to the recognized cross-antigenicity between both viruses, reactivity was attributed to WEE epizootic virus. No viral activity was detected in the domestic birds. With this contribution, the study of one epizootic cycle of WEE virus was completed: pre-epizootic silence (1977-1980), epizootic (1982-1983) and residual focus plus inapparent infections during a post-epizootic period (1983-1986).

Transmission of western equine encephalomyelitis virus by Argentine Aedes albifasciatus (Diptera: Culicidae).

Aedes albifasciatus (Macquart) has been considered a potential vector of western equine encephalitis (WEE) virus in Argentina because it has been found naturally infected, it is susceptible to infection per os, and its distribution and feeding habits are compatible with those of an epizootic and epidemic vector. Ae. albifasciatus females collected in the vicinity of Cordoba, Argentina, were highly susceptible to WEE virus perorally (ID50 less than 0.5 Vero cell plaque-forming units) when fed on viremic chicks. Virus transmission trials were done 9 to 16 d after infection by feeding potentially infected mosquitoes individually on chicks. Among 31 mosquitoes that survived the incubation period, six refed on susceptible chicks. Virus transmission by bite was demonstrated by five of six (83%) mosquitoes. Therefore, the final piece of evidence incriminating Ae. albifasciatus as a natural vector of WEE virus has been obtained. This is the first mosquito species to be incriminated as a vector of WEE virus in South America.

Experimental neuroinvasiveness of wild and laboratory Junin virus strains.

The neuroinvasiveness of Candid 1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus (Cm), of different ages inoculated by a non-neural route. Infectivity in brain, blood and organs, as well as lethality, were determined. The results with the 3 hosts indicate that Junin virus neuroinvasiveness is virus-strain-dependent, host species- and age-dependent, with the Candid 1 strain proving to be the least neuroinvasive of the strains studied. The lethal efficiency index (log PFU/LD50) in 2-day old albino mice and the neuroinvasiveness index (Log PFU/ND50) in 6 +/- 1 day-old Cm of the various strains using the intraperitoneal (ip) route could therefore be useful markers of Junin virus neuroinvasiveness. Moreover, different patterns of infection were established using the results of the presence of infectious virus in brain and viraemia in the 3 hosts. In nearly all cases, virus neuroinvasion was present without detectable viraemia (virus in plasma). Current evidence leads to the assumption that virus might reach the brain associated with the white cells in blood (undetectable by conventional isolation methods) or by another possible mechanism of neuroinvasion which is not haematogenous.

Rapid vascular clearance of two strains of Junin virus in Calomys musculinus: selective macrophage clearance.

Clearance of Junin (JUN) virus strains with different virulence for Calomys musculinus (Cm) was followed using the Candid #1 virulent and CbaFHA 5069 attenuated strains. In addition, virulent virus albino mice (AM) were included as control host and Venezuelan equine encephalitis (VEE-VI) virus as control virus. The virus inoculum (Vo) and the blood samples (Vt) obtained at different times post-inoculation (p.i.) were titrated on Vero cells and the cleared plaque forming-units (PFU) were calculated as the log Vt/Vo. In Cm both JUN virus strains were cleared rapidly (within 5 min the Candid #1 strain and within 10 min the CbaFHA 5069 strain); meanwhile, VEE-VI virus could be recovered from blood until 30 min p. i. Furthermore, JUN and VEE-VI viruses showed the same behaviour in Am as in Cm. We conclude that the JUN virus strains of different virulence for Cm did not show differences in their clearance from the blood of these animals. Moreover, the rapid clearance observed was independent of the animal host and viral dose.

Localized eastern equine encephalitis in Santiago del Estero Province, Argentina, without human infection.

In 1981, a localized epizootic of Eastern Equine Encephalitis (EEE) occurred in irrigated areas of four counties in the province of Santiago del Estero, Argentina. The diagnosis was confirmed by serology, and there was no evidence of involvement of Western or Venezuelan equine encephalitis viruses. The overall incidence of equine encephalitis was estimated 17%, the case-fatality rate at 61% and the inapparent: apparent infection ratio less than or equal to 2.9:1. This is the first localized epizootic defined in Argentina and the first in which EEE has been found as the sole etiologic arbovirus. This posed the possibility to look for human infection in the area. In spite of a careful surveillance, no evidence of human disease or infection was found, differing from the situation in USA where EEE virus is a public health problem. Nevertheless vector/s and vertebrate hosts involved in the transmission cycle in Argentina remain unknown, precluding at present speculations on the potential human risk.