Legionnaires Disease,Legionella,Legionellosis

INDEX:
I PRESENTATION
II INTRODUCTION
111 METHODOLOGICAL ASPECTS

III.1-Definition of cases
III.2-Encuesta case and analysis diagnostics
III.3-Tecnicas epidemiologist of laboratory

IV. DESCRIPTIVE STUDY OF THE OUTBREAK
IV. 1.-Number of cases -
IV. 2.-Manifestaciones clinical -
IV. 3.-Evolucio'n of the -
IV. 4.-Caracteristicas cases of temporo space -
IV. 5.-Distribution person -
V. - MICROBIOLOGICAL RESULTS
VI. - ENVIRONMENTAL INVESTIGATION
VII. - ANALYSIS EPIDEMIOLOGIST
VIII MEASURES OF CONTROL

IX. - CONCLUSIONS X BIBLIOGRAPHY
XI. - ANNEXED: Preventive Actions Forehead To New Outbreaks of Legionella

The handling and management of an epidemic outbreak of legionellosis, like which it took place in the months of September and October of 1996 in Alcala de Henares, are always complex, and require to establish a solid and complex strategy of coordination between professionals, non sanitary toilets and, specialized in different disciplines from the scientific knowledge (clinical, microbiologists, epidemiologists, industrial professionals of Environmental Salud, engineers and others). But in addition, tratandose of a outbreak of legionellosis opened in the community, with the consequent difficulties of location and control of the center origin of the same one, that affected an elevated number of people, with the social impact that it entails, does that the effort to develop by the health institutions is still more considerable. In the management of this epidemic outbreak, that fundamentally affected a zone of Alcala de Henares of 46,000 inhabitants, the administrations were implied Local, Autonomic and Central:

The City council of Alcala de Henares, person in charge of whatever affects to the health between its citizens.

Social Consejeria of Health and Services of the Community of Madrid as Sanitary Administration Regional person in charge of the investigation of the outbreak and to dictate and to control the application of the preventive measures of Public Salud for its eradication.

The Hospital Prince of Asturias of INSALUD, pertaining to the Ministry of Health and Consumo, person in charge of the sanitary attention of the patients.
Institute de Salad Carlos III, also pertaining to the Ministry of Health and Consumo, through its National Centro of Microbiology, like Central Laboratory of support to National Sistema of Salud.
With the purpose of reinforcing the mechanisms of institutional coordination and unifying the messages directed to the inhabitants of Alcala de Henares and to means of communication, in Consejeria de Social Health and Services a Committee of Experts constituted itself, with representatives of the three implied administrations and of Scientific Societies, that made valuable contributions in the management of the outbreak. The composition of this Committee was the following one:

- President of the Committee:
*Dna. Rosa Posada Chapado (Advisor of Health and Social Servicios)

- Institutional Representation: -
*D. Bartholomew Gonzalez Jime'nez (Mayor of Alcala de Henares)

*D. Jose Luis Perez de Rueda (Advisor of General Director of Instituto de Salud Carlos III)

*D. Emilio Bable Santiago (Head of Service of Microbiology of the Hospital Gregorio Mara6no'n) -

*D. Carlos Jorge Domingo Fernandez (Director of National Center of Microbiology)

*D. Alvaro the Olive Lozanos (Head of Service de Microbiology Diagnostic of National Centro of Epidemiology)

*D. Jose Manuel Echevarria May (Head of Service of Orientacion Diagnostic of National Centro of Microbiology

Dna. Carmen Pelaz Antolin (Responsible for the Laboratory of Legionella of National Center of Microbiology)

D. Fernando de Ory Mancho'n (Head of Section of Microbiology Diagnostic of National Centro of Microbiologoy)

*Dna. Cecilia Seaable Seine fishingable (Head of Area of Investigation of National Center of Microbiology)

*D. Felipe Vable Herranz (General Director of Prevention and Promotion of Salud of Consejeria de Sanidad and Social Services)

The present report must as objective present/display investigations made during the outbreak, directed to know its origin and eliminate able, and the results of the same ones, like final basic document, support of possible scientific developments that in the future can take place in their different slopes, epidemiologist, microbiological clinic and. In him all the sanitary professionals responsible for such investigations have participated, and constitutes in addition the report that on the outbreak they emit, to common agreement, the implied administrations.

Day 11 of September of 1996 the sanitary services of the INSALUD of Alcala de Henares notified Servicio de Public Salud of Area 3, of Consejeria of Social Hanidad and Services, an increase on the habitual thing of the extra hospital numbers of pneumonia taken care of in the Hospital Prince of Asturias in the two previous weeks. One was pneumonia that responded favourably to the treatment with eritromicina and not with other guidelines of antibiotic treatment. Immediately, from Service of Public Health of Area 3, an equipment of epidemiologists initiated the investigation epidemiologist, in narrow collaboration with the sanitary professionals of the Hospital Prince of Asturias and Primary Atencion of the municipality, with the purpose of confirming the existence of an epidemic outbreak and sustaining a work hypothesis that as far as possible allowed the adoption of measures of control of that one.

At the same time, the opportune devices of monitoring articulated epidemiologist at local and regional level. Thanks to the computerized registry of Service of admission de emergencies of the Hospital, could be confirmed, comparing the present cases with the taken care of ones and entering of previous years, the existence of a outbreak of pneumonia and a space grouping of cases in the North zone of Alcala de Henares. Cases identified retrospectively of pneumonia taken care of and entere in Hospital from 25 of August (date in which the manifest beginning of the outbreak was demonstrated) and a survey was designed and applied to these cases oriented epidemiologist to describe the outbreak and to identify the common to cases and associated factors of risk to pictures of pneumonica pulmonary affectation. The results of this survey revealed the following excellent findings: aggregation of cases in the average labour relative did not exist nor; common exhibition to closed places of public concurrence did not exist; there were no common antecedents of exhibition to animals; they did not exist antecedent of food ingestion of suspicious origin (sanitarily not controlled) or unusual in the habitual diet of the cases; indications of implication of some toxic agent did not exist either; the average age of the affected ones was of 68 years. The previous findings, next to the clinical evidences and the good answer to the antibiotic treatment with eritromicina, oriented the etiological hypothesis towards an infectious outbreak of pneumonia in the community, probably due to Legionella. At that same moment the environmental investigation began, directed to locate emitting centers of aerosols contaminated by Legionella. Immediately after known the first results microbiological that guaranteed the legionellosis hypothesis, the appropriate preventive measures for the population of Alcala de Henares were started. Later, the etiologic hypothesis of a outbreak of pneumonia by Legionella definitively was confirmed by new microbiological results. The cases studied in this report correspond to all those that, fulfilling the case definitions which sanitary Prince of Asturias and other centers between the 25 of August settle down themselves, were taken care of in the Hospital and the 26 of October of 1996 (date of sanitary attention of the last case including in the outbreak). After these dates the opportune devices of monitoring stayed in alert (and they are continued maintaining at the present time) epidemiologist, without new cases related to the outbreak were detected.

With the purpose of defining the cases to include in the outbreak a classification of the same ones in three categories has settled down: suspicious, probable and confirmed cases. This classification has been made taking care of a clinical criterion and microbiological criteria and epidemiologists. Clinical criterion: It is constituted by those clinical premises including in Provisional Operative Definition of case handled by the Hospital Prince of Asturias during the pursuit of the outbreak, once established highly probable the etiological hypothesis of legionellosis. This criterion includes:
Greater criteria:
1. Time of evolution of the symptoms inferior to 10 days.
2. - Radiological infiltrated presence of pulmonary.

Smaller criteria:
(a)- Fever.
(b) - general Malaise, artromialgias or migraine.
(c) - respiratory Symptoms: cough, giveable or pleuritico pain.
(d) - alveolo-arterial Gradient superior to 20 mmable.

Criteria of exclusion: · Positive Baciloscopia in respiratory secretions. · * Bacterial isolation in blood, pleural liquid or secretion bronchial, different from Legionella. · * High suspicion of broncoaspiracion. · *Diagnosis of another specific disease that could justify the symptoms. For the fulfilment of the clinical criterion the fulfilment of the greater criteria is required, of three of smaller the criteria and the absence of all the criteria of exclusion.

Microbiological criteria: wo types of microbiological criteria settle down: Criteria of confirmation:
1.-Aislamiento de Legionella in biological samples.
2. - Demonstration of a quadruple or greater increase of the title of antibodies as opposed to Legionella pneumophila SG1, until reaching 1/128 more or.
3. - Demonstration of a title of antibodies as opposed to Legionella pneumophila SG1 ³1/256

Suggestive criteria:
a) - Detection of antigen of Legionella pneumophila SG1 in orable by enzimoinmunoensayo.
b) - Detection of Legionella pneumophila SG1 in biological samples by means of enzymatic amplification of DNA (PCR).
c) - Demonstration of a quadruple or greater increase of the title of antibodies as opposed to Legionella pneumophila SG1, up to 1/64. d) - Demonstration of a maintained title of antibodies as opposed to Legionella pneumophila SG1 of 1/128.

Criteria epidemiologists: Initially, Provisional Operative Definition of Cases included the criterion in the last residence epidemiologist month in Alcala de Henares. With it was tried that the definition was highly sensible.
Later, as the high percentage of cases with microbiological criteria of confirmation was observed that lived or been recently had exposed to the North zone of Alcala (97.63%), was decided, with the purpose of increasing to the specificity of the definition, maintaining the high sensitivity, making the criterion more restrictive epidemiologist of exhibition and to reclasifition the cases according to the same one. On the basis of the previous thing, the following criteria settled down epidemiologists who now we maintain:
1. - To reside habitually in the North zone of Alcala that includes/understands municipal districts 7, 8 and sections censusable 4, 5, 7, 8, 14 and 15 of district 6.
2. To have visited at some moment, during the three previous weeks in the beginning of the disease, the North zone of described Alcala.
3. According to these criteria they are distinguished:

All case that, having received medical assistance between days 25 of August and 26 of October of 1996, the criterion fulfils clinical and one of the epidemiologists.

CASES PROBABLE:
All suspicious case that it fulfils one or more of the suggestive microbiological criteria.

CASES CONFIRMED:
All suspicious or probable case that it fulfils one or more of the microbiological criteria of confirmation.

After the notification to Service of Public Salud of Area III of Consejeria of Health and Social Services, on the part of the services of medical assistance of the INSALUD of Alcala de Henares, of the cases that motivated the suspicion of the outbreak, information of the computerized registry of Service of Admission of Emergencies of the Hospital was successfully obtained Prince of Asturias, that included name and last names of the taken care of cases, date of attention toilet, date of birth, I domicile, telephone, initial diagnosis and destiny of the patient.
In agreement with this information, that it allowed to identify initially to the possible cases taken care of by the hospital, it was designed and it made one first survey oriented epidemiologist to describe the outbreak and to identify the common to cases and associated factors of risk to pictures of pneumonic pulmonary affectation: animal trips, exhibition, exhibition to places public, ingestion of unusual foods or origin non controlled sanitarily and exhibition to other well-known cases.
Later, once known the first positive results serological Legionella, that guaranteed the initial hypothesis of a legionellosis outbreak, one second oriented survey was designed fundamentally to investigate the exhibition of the cases to possible sources of formation of aerosols, as much in the home as in the public route: use of the shower in the home, centralized sanitary hot water, routine strolls, exhibition to places closed public, etc. Also the intense aggregation of cases in the same one was asked specifically for the exhibition the zone of Alcala de Henares considered like zone of risk, given North: zone inhabited to the north of Complutensian Vi'a that includes/understands municipal districts 7, 8 and sections censusable 6-4, 6-5, 6-7, 6-8, 6-14 and 6-15, all of them of district 6.
This second survey was made to all the possible cases that were appearing during the outbreak, either by telephone, or personally when the patients remained entered, not knowing the encuestadores the classification of each patient at the moment for making the survey, when not having then the microbiological information of those. With the information gathered in this survey the study of the environmental factors of risk was carried out, valuing the exhibition to the same ones in a period of three weeks before the beginning of the symptoms.
The studied factors of risk were: exhibition to the zone of risk, exhibition to the contaminated cooling towers with the same stock of Legionella detected in the patients, exhibition to the shower in the home, existence in sanitary hot the water house centralized and existence in the building of the house of potable water tank. In order to study the exhibition to the cooling towers an influence area was defined for each one of them, locating the tower in the center of a circle of 200 meters of radio, in agreement with the established thing in similar studies, and recodifition the database valuing itself for each patient the exhibition to this area, considering the address of the patients and the routes followed in the made strolls the three previous weeks in the beginning of the disease (routine or precise strolls to go to concrete places or by reason for leisure). The analysis of the environmental factors of risk was made by means of two complementary designs:
1. - I design
1: A study of cases and controls with the following definitions has been made:

*Cases: All patient taken care of in a sanitary center from the 25 of August the 26 of October of 1996, resident one in Alcala de Henares or that has visited the city during the three previous weeks in the beginning of its disease, with clinical diagnosis of pneumonia and microbiological criterion of confirmation of recent infection by Legionella.
*Control All patient taken care of in a health center from the 25th of August the 26th of October of 1996, resident one in Alcala de Henares or that he has visited the city during the three previous weeks in the beginning of its disease, with clinical diagnosis pneumonia (excluded those from well-known cause different from Legionella) and without microbiological criterion of confirmation of recent infection by Legionella.
2. - I design
2: A study of cases and controls with the following definitions has been made: *Case: All patient who was entered in the Hospital Prince of Asturias the 8 of October of 1996, resident in Alcala de Henares, with clinical diagnosis of pneumonia according to Provisional Operative Definition of case (possible case of legionellosis), excluding the cases with stays superior to 15 days before the 8 from October. *Control: All patient entered the Hospital Prince of Asturias 15 days before or after the 8 of October, by cause different from pneumonia, excluding all respiratory pathology or febrile process 30 days before the entrance, and resident in Alcala de Henares. controls have mated by sex and age (±5 years).
2 controls by case have been chosen. In the analysis of both designs Ratios with intervals of different species and serogroups from Legionella, and those isolated ones identified like L. pneumophila SG1 were sub-group able with the standardized panel of monoclonales antibodies, who allows his classification in three greater sub-groups, Pontiac, Olda and Bellingham, and ten smaller sub-groups.
3.-Detection of Legionella by means of direct inmunofluorescencia in samples of beable, respiratory secretions obtained by broncoscopia and pulmonary weave, using commercial preparing and following indications of the manufacturer.
4. - Detection of antigen of L. pneumophila SG1 in orable by means of enzimo-inmunoensayo, using commercial preparing and putting under some of the samples to a technique of selective ultrafiltration.

5.-Detection of genomic material of Legionella pneumophila by means of enzymatic amplification of DNA (PCR), using the " Enviro Amp " Legionella kit. In addition to the diagnosticas tests for legionellosis, to all the cases of protocolizada form the following studies were also made to them:
Serologroup A)-Estudio of antibodies as opposed to influenza To, influenza B, adenovirus, sincitial respiratory virus, Mycoplasma pneumoniae, Coxiella burnetti phase II and Chlamydia psittaci, by fixation of the complement, according to a standard procedure.

b)-Hemocultivo for different pathogens, excluyen dose the cases with positive hemocultivo different from Legionella.

C)-Estudio of isolation in different types from cellular cultures for the detection of viral agents

D)-Estudio of electronic microscopy for search of different Pathogens. For the culture of Legionella from the samples of environmental origin, these were seeded, after their concentration and decontaminacio'n 4,5, in means of culture BABLE and MWY. The subgrupado identification and of the isolated ones was made as it has been detailed previously for the clinical samples.

DESCRIPTIVE STUDY OF THE OUTBREAK Nº of cases
Table 1 presents/displays the cases including in the outbreak, in agreement with the criteria of established, distributed inclusion of case according to its classification in certainty degrees and confidence to 95% like measurement of the effect calculated Odds. In design 2 the calculations for matched up data have been made. The estimations were made by stratified analysis and logistic regression in design 1 and by bivariant analysis in design 2. For all the calculations of rates of population incidence the data of Municipal Padro'n of Alcala de Henares updated to November of 1996 have been used. Diagnosticas techniques of laboratory The used techniques of laboratory for the legionellosis diagnosis were the following ones:
1.-Deteccio'n of antibodies as opposed to Legionella pneumophila SG1 by IFI, according to technique used by the microbiology laboratory.
2.-Aislamiento and identification of the bacterium by means of culture of samples of respiratory secretions (esputos and samples obtained by broncoscopia), woven of autopsy and pulmonary biopsies. The culture of the bacterium was carried out by means of direct inoculation in means of culture BABLE and BMPA in the case of samples noncontaminated and in BABLE, after a process of decontamination, in thre case of the esputos. The identification of the isolated ones was made as opposed to by means of IFI sanitary centers in which they were attended, being these those that notified and informed the cases.
Although from Primary Attention more cases were notified, in the single corresponding column have been reflected the cases taken care of exclusively in that attendable level, incluyen dose in the one of the Hospital Prince of Asturias the cases derived to the same one for their attendance. If we excluded from the total of cases 3 that they did not have habitual residence in Alcala de Henares, a rate is from incidence for all the population of this municipality of 132 cases by 100,000 inhabitants.

The existing discrepancy between the total number of cases taken care of by the Hospital Prince of Asturias that when finalizing the outbreak occurred like official (262 cases) and the present number (214 cases), must to that that one, being provisional, has been corrected when applying the definitive criteria of inclusion of case, descartadose 48 patients that when not fulfilling such criteria cannot be considered as cases of the outbreak

Table 1. - Distribution of cases according to its classification and centers of medical assistance.

Classification of cases	Hospital Prince of Asturias	Primary Attention of Alcala de Henares	Other Hospitals	Total
Confirmed	86	4	2	92
Probable	42			42
suspects	86	4		90
Total	214	8	2	224

In this section the excellent clinical data of all the cases taken care of in the Hospital appear Prince of Asturias.

- Fever 94% - General Malaise 78% - Cough 47% - Giveable 42% - Artromialgias 41% - Migraine 34% - Pain Pleuri'tico 25%	Expectoracio'n 23% Nauseas 19% Hemoptisis 6% Confusion 6% Dayable 4% Agitation 2% Other 16%

- objetivada Fever 91% - Crepitantes 64% - Sibilancias 14% - Youable 9% - Cyanosis 6%	- Hepatomegalia 4% - Adenopati'as 1% - Haveable Theable 1% - Ofable Eruption. 0% - Other 8%

Between the analytical data of interest it is possible to emphasize the following ones:

- Anemia (Hb - Leukocyteable - Creatinina - Hiponatremia - Ldh - Ggt - F. Alcalina	<11gr%) 11% (>15.000/mm3) 18% (>2mg/dl) 8% (Na<130mOsm/L) 6% (>500 Ui/l) 23% - Gpt (>100 Ui/l) 5% (>100 Ui/l) 16% (>300ui/l) 18%

The average number of leukocytes was 11.850 able and the average value of mable creatinina 1,29.

The gasometricos analyses revealed the following results:
- pO2>45MMHg 6%
- pO2<65mmHg 55%
- pO2<60mmHg 36%
- Gradient Alveolo-arterial>45 17%
- Gradient Alveolo-arterial>60 2%

The average values of pO2, pO2 and of alveolo-arterial Gradiente were respectively of 37.84mmHg, 63.53mmHg and 34.55.
The radiological data more substitutes were the following ones:
- Radiological Pattern: to
*Alveolar 89%
*Intersticial 6%
*Mixto 6%
- Radiological Extension: to *Unilobar 82%

*Multilobar able. 8% to
*Multilobar bable. 10%
- Radiological Affectation: inferior *Lobulos 67%
(izdo. 51%, dcho. 49%)
*Lobulos superior 38% (izdo. 45%, dcho. 55%)
*Lobulo means 13%
- pleural Spill:
*Sin spills 89% *Unilateral 10%
*Bilateral 1%

Evolution of the cases
From the moment of the beginning of the symptoms to the one of the attendance in the Hospital Prince of Asturias 4,3 days passed like average. The clinical improvement after the treatment also took place in 4.3 days like average, being good in general the macroidos answer to the treatment with. Objectiveable three reingresos in hospitalization: one of them, who evolved favourably, with pathology related not clearly to the pneumonico process, a second case of 92 years that passed away with suspicion of added broncoaspiracion and a third case of 70 years that reingreso in septica situation and I fail multiorganic, passing away later by secondary septico shock to pseudomembranosa colitis, considera dose like a complication of the antibiotic treatment.
Of the 224 cases included in the outbreak, 9 passed away, which throws a rate of 4% lethality, habitually referred inferior number to for these outbreaks. The average age of the deceaseds was of 71,2 years (rank 41-91). All of them presented/displayed significant pluripatologi. The discrepancy between the present number of deceaseds and the one that occurred as official when finalizing the outbreak (15 deceaseds) also obeys to that this one last, provisional one, she has been corrected when applying the definitive criteria of inclusion of case, descartadose of the cases of the outbreak 6 deceaseds who did not fulfil such criteria.
Characteristics of person
The average age of the total of cases was of 59,3 (s=18.3), being 62,7 (s=13) for the confirmed cases, 60 (s=16.7) for probable cases and 55,4 (s=22.5) for the suspicious cases. The analysis of the variance of these three averages reveals a significant difference (p<0.05) between the average of confirmed cases and the one of suspects.
Figure 1 shows the specific rates by age for the total of cases and by sexes. It can clearly estimable that the incidence increases progressively as of the 54 years and that the age group more affected is the one of 70-79 years. The distribution is similar for men and women, being the rates of these inferior ones in all the affected groups of age more, safe in the one of 70-74, in which he is slightly superior to the one of men.
Figure 1. - Rates of incidence you specify by age and sex for the total of cases.

The 58,5% of the total of cases were men and the 41,5% women, being the specific rates by sex, standardized by age and for all the population of Alcala de Henares, 164,9 by 100,000 inhabitants for men and 103,9 for women, and the masculineable reason of 1.59. If we analyzed the masculineable reason of for cases separately confirmed, probable and suspicious, we observed that for the confirmed cases the reason descends to 1,15, being 1,8 and 2 for the probable and suspicious cases, respectively.
In addition to the age average outpost, the registered more important factors of risk between the antecedents of the cases were: $able - Tabaquismo 32% - Hepatopati'a 5% - EPOC 23% - Inmunosupresion nonVIH 1,3% - Cardiopathy 22% - Other 23% Alcoholism 10% In relation to the professional activity of the cases the most frequent groups were Workings domestic (24.2%) and Retired (18.8%).

Figure 2 shows the temporary distribution of all the cases and the cases confirmed, according to date of beginning of symptoms.
In 17 cases the date could not be known beginning of symptoms, reason why they are not represented in the figure. It can esteemable which the outbreak stops the total of cases seems to present/display two phases of different intensity, being second, between days 15 of September and 5 of October, of greater intensity. Nevertheless, when single considering the confirmed cases the appearance of the same ones is more homogenous in the time. The last confirmed case began with symptoms on day 11 of October.

Figure 2.
Temporary distribution of the total of cases and the confirmed cases, according to date of beginning of symptoms.

From the beginning of the investigation epidemiologist of the outbreak a clear space aggregation of cases in the North zone of the municipality was observed, in agreement with its direction of residence.
For objectable such aggregation the rates of censusable incidence by sections of Alcala de Henares have calculated, standardized by age.

Figure nº 3 shows the graphical representation of these rates for all pneumonia studied during the outbreak, with microbiological criteria of confirmation for Legionella. In her it can observeable how the sections censusable with elevated rates of incidence more are located in the North zone of the municipality.
Since in addition, all the patients with pneumonia and microbiological criterion of confirmation who lived outside the North zone, been had exposed to that zone in the three previous weeks in the beginning of his disease, with the exception of two, he was decided to delimit this North zone as zone of risk and of including the exhibition to the same one as criterion epidemiologist of inclusion of case.

Figure 3.
Rates of incidence of pneumonia with microbiological criterion of censusable confirmation po sections, standardized by age

Figure 4 shows the rates of censusable incidence by sections for all the cases incluidos in the outbreak.
The distribution of cases is similar to the one of previous Figure. The sections censusable with elevated rates of incidence more are located grouped in a zone between Complutensian Via, the line of the railroad and the O´Donnell Park.

Figure 4.
Rates of incidence of total cases of the outbreakof censusable legionellosis by sections, standardized by age

In this section the microbiological results of the studies appear on human samples. In the section corresponding to the environmental investigation the microbiological results of the analyzed water samples will appear. In the studies of isolation of Legionella 9 isolated of L.s pneumophila SG1, samples coming from 7 cases recovered: 7 samples, coming from 5 patients, obtained by broncoscopia, 1 of beable one and 1 of pulmonary weave coming from autopsy. All of them belonged to the sub-group greater Pontiac, sub-group smaller Knoxville. Of the 7 cases with isolation of Legionella, 4 agree with serolo'gicos data diagnoses (one of them in addition with positive positive PCR and 2 with antigenuria) 1 with suggestive serologicos data (seroconversion to 1/64), 1 with positive negative serologicos data and PCR, and one rest in which it was not had another type of sample. In the serologicos studies the following positive or suggestive results of recent infection by L. were obtained pneumophila: Seroconversion to ³1/128 49 cases - Seroconversion to 1/64 10 cases - Title unique or maintained ³1/256 40 cases - maintained Title =1/128 15 cases In the studies of detection of antigen of L. pneumophila in orable positive results in 37 cases were obtained. In 23 of them also positive results by other diagnosticas techniques (isolation, serologia or PCR) and in the 14 rest were obtained antigenuria was the only diagnostica test of laboratory. The tests of detection of genomic material by enzymatic amplification of DNA gave positive results in 12 cases. In 9 of them also positive results by other diagnosticas techniques were obtained and in the 3 rest the technique of the PCR was the unique one that showed a positive result.
All the samples analyzed with the technique of direct immunofluorescence gave negative results.

After the first clinical data and epidemiologists made the hypothesis probable of a legionellosis outbreak, the environmental investigation began. Given evident to the space grouping of cases in the North zone of the municipality and the absence of common exhibition from those to closed places of public concurrence, the environmental investigation went fundamentally to the location in this zone of potential emitting centers of Legionella, able to emit aerosols to the public route, and the detection in same of the stock the cause of the outbreak. Also one studied the potable water network of the zone, as well as other facilities of storage and water distribution for public use (centralized sanitary hot water, wells, etc.), with the purpose of identifying, to where outside possible, the origin of the outbreak and in general the presence of environmental factors of risk which they could contribute to explain that one.
Table 2 presents/displays all the water samples taken in the investigation from the outbreak, according to its the positive origin, as well as number of studied facilities and results for samples and facilities. A total of 109 samples coming from 85 different facilities was taken, being positive to Legionella 46% of the samples and 45% of the facilities. More the frequently contaminated facilities were the cooling towers, detectandose presence of Legionella in a 63% of the same ones, and the water tanks of communities of neighbours (5 contaminated deposits of 7 studied, 71%).

**Table 2. Isolations** **of Legionella in environmental samples of Alcala de Henares**
Origin of the samples	Nº samples / Nº facilities	positive Samples / positive Facilities
Tower Refrigeration	63/41	38/26
Sanitary Water Diffusing Deposit	17/15 7/7	4/4 5/5
Terminal network	4/4	1/1
washing cars	5/5	0
Well	6/6	1/1
Tanker	2/2	1/1
Source	1/1	0
Other	4/4	0
Total	109/85	50/38

Table 2. Isolations of Legionella in environmental samples of Alcala de Henares

In Table 3 are the results of the identification of the isolated ones. From the 50 water samples with positive isolation isolated ones of Legionella were obtained 53, since in three cooling towers two different species or serogroups were identified. The 53 isolated ones were identified as it follows:
49 (92.5%) of species L. pneumophila
4 of different species (2 L.s micdadei, 1 L. bozemanii / L. longbeachae SG2, and 1 Legionella sp).
Of the 49 isolated of L. pneumophila, 42 (86%) belonged to the SG1, and the 7 rest to other different serogroups. Of the 42 isolated of L pneumophila SG1, 12 belonged to the sub-group greater Pontiac (8 to the sub-group smaller Knoxville and 4 to the sub-group smaller Benidorm) and the 30 rest belonged to the sub-group greater Olda, all of them of the sub-group smaller Oxford/Camperdown.

Table 3. Distribution of the isolated ones in species of Legionable, serogrupos of L. pneumophila and sub-groups of L. pneumophila Sg 1.

Figure 5
presents/displays the results obtained in first samples (excluding second and successive samples) according to its origin, detallandose the negative results and the distribution of the isolated ones by species, serogroups and sub-group.
From 85 first samples they were positive to Legionella 37 (43.5%). The cooling towers were positive in 61% of the samples.

Figure 5: Results of isolation of Legionella in first samples according to his origin

Those isolated ones identified like L. pneumophila SG1, sub-group greater Pontiac, sub-group smaller Knoxville (9 of human origin and 8 of environmental origin:
4 cooling towers and 2 deposits of communities of neighbours) were studied by the techniques of molecular Biology of chain reaction of polymerase (PCR arbitrary) and electroforesis in field pressed after digestion of the DNA with two enzymes of restriction, Not I and Sfi I. Both techniques demonstrated the identity of the two groups of isolated.

Figure 6 shows the location in Alcala de Henares of the 4 cooling towers and the 2 water tanks contaminated with L. pneumophila SG1, sub-group greater Pontiac, sub-group smaller Knoxville. The tower number 1 and the two deposits are located in the zone of maximum incidence of the disease.

Figure 6. Location in Alcala de Henares of the cooling towers and water tanks contaminated by L. pneumophila SG1, greater sub-group
Pontiac, sub-group smaller Knoxville

The study of the potable water network, specially of the North zone, showed the presence of important sanitary, structural deficiencies and functional, that drew trasfondo I cause to explain the contamination of the cooling towers: Presence of numerous water tanks of Communities of Vecinos (more than 2000 in Alcala de Henares and 719 in the North zone) that, generally, had deficient conditions of cleaning and, given its characteristics, they cause retentions of the water during periods prolonged of time and ebb tides to the network. - Presence of numerous terminal points of network (13 in the North zone), some of them with low or null values of chlorine after the superchlorination of the network and drained presence of turbidity and dirt to the being manual Chlorine in some of the municipal deposits (deposits of Meco). nonhomogenous Distribution of chlorine in the network (important oscillations of the free residual chlorine level in different points from the network).

ANALYSIS EPIDEMIOLOGIST
According to one commented in the methodology section, two complementary types of study with the purpose of establishing associations between the exhibition to diverse environmental factors of risk and the fact were designed of suffering one pneumonia by Legionella.

Table 4
presents/displays the results of the analysis of design 1 (cases of pneumonia with microbiological criterion of confirmation for Legionella as opposed to the rest of pneumonia like controls). Or for each variable have calculated fit by sex and age by means of logistic regression.
A statistically significant association between living in the delimited zone is demonstrated as on visitable risk or and to be case, OR=15.7 (3,7-66.2).
When analysing the exhibition to the cooling towers, is a statistically significant association between exhibition to tower 1 and to be case, OR=3.14 (1.8-5.5), and between the exhibition to tower 4 and to be case, OR=2.02 (1.2-3.5).
Analysing only the visits to the circles of the cases that live outside them, they continue being significant the associations between being case and of visiting tower 1, OR=3.48 (1.9-6.5), or of visiting tower 4, OR=2.04 (1.2-3.6). Or, fit by age have calculated in addition and sex, of the exhibition to I circulate 1 and to I circulate 4, controlling the possible effect of the interaction between both, manteniendose the associations commented previously, with values of Or for tower 1 of 3.95 (1,90-8,23) and for tower 4 of 2.85 (1,12-7,29).
Significant associations for the rest of studied factors of environmental risk have not been detected.

TABLE EXHIBITION WITH ISOLATION OF L. PNEUMOPHILA SG1 SUB-GROUP GREATER PONTIAC SUB-GROUP SMALLER KNOXVILLE

Table 5
presents/displays the results of design 2 (cases of pneumonia with clinical criteria of inclusion in the outbreak, entered 8 of October, as opposed to cases entered next dates with diagnosis different from pneumonia).
In this study significant association between living in the zone of visitable risk or and to be case OR=6,25 (2,06-25,5), and between the exhibition to tower 1 has also been demonstrated and to be case OR=5.18 (1.76-18.45). Significant association with the exhibition to tower 4 has not been demonstrated nevertheless, although the association is in the limit of the meaning, nor with other factors of risk.

Like complementary approach to the commented designs, 100 and 200 meters circles of and 200 and 300 meter calculated in addition the gross rates to population incidence of pneumonia with microbiological criteria of confirmation for Legionella in a radius of 100 meters around the four towers and in the sectors between.
Table 6 shows the results of these calculations. It can esteemable that around tower 1 occurs the elevated rates of incidence more and than a decreasing gradient of incidence exists as we moved away of the same one, which does not happen in the other towers.

Table 6.
Incidence of cases of neumoni'a with microbiological criteria of confirmation around
the contaminated cooling towers with L.pneumophila SG1, sub-group greater Pontiac, sub-group smaller Knoxville

The results reviewed epidemiologists, together with the microbiological or commented data, indicate as origin of the outbreak to one or several cooling towers. These facilities have been cause of outbreaks of the same nature in numerous occasions, according to gathers scientific Literature

Day 23 of September communicated by telephone to Centro de Public Salud of Alcala de Henares, on the part of National Center of Microbiology, the finding of 5 high titles (³1/256) of antibodies and one seroconversion to Legionella pneumophila SG1, in patients entered by pneumonia in the Hospital Prince of Asturias.
Since, in addition, these findings agreed with the most probable hypothesis that until that date they sustained to the clinical data and epidemiologists, it was decided as opposed to to dictate immediately preventive measures for the population of Alcala de Henares legionellosis.
The first measures were of general character, destined to avoid the formation of aerosols in the home (to avoid the shower and the domestic pulverization of water) and in places public (to avoid the irrigation by aspersion, the irrigation of streets in hours of public concurrence, to paralyse the ornamental operation of sources and the one of systems of refrigeration of water and air conditioning by water...), to the time that began the active search, in the North zone of Alcala de Henares, device and cooling towers, in general, or system of refrigeration and air conditioning that could cause aerosols. These measures were spread through a municipal side.
The superchlorination of the network was carried out until reaching a minimum of 3 ppm of residual free chlorine in all the points of that one, during a non inferior time to 8 hours. Later the chlorine stayed around 2 ppm until the remission of the outbreak. The cleaning of the cooling towers of the North zone of Alcala de Henares determined day 26th of September. Since census of the same ones did not exist, it was necessary to make an exhaustive active search, identification of 23 towers, that were cleaned and disinfected or sealed between days 30th of September and 7th of October. Between such towers were the identified ones in this report like towers 1, 2, 3 and 4, contaminating with L. pneumophila SG1, sub-group greater Pontiac, sub-group smaller Knoxville.
Between days 10 and 12 of October one second cleaning of the towers by specially qualified companies was carried out, like additional guarantee.

The purge and cleaning of terminal points of the potable water network (blind points) and the cleaning and disinfection of water tanks of Communities of Vecinos, were carried out after knowing the existence these points of risk in the municipal network.
The cleaning of sanitary hot water tanks centralized and the extension of the cleaning of water tanks and cooling towers to all the municipality, were determined in later dates also like measures of additional guarantee.
According to it can estimable in Figure 7 these measures of control were adopted in early dates, the great majority of them before definitive the etiologica confirmation of the outbreak by the Committee of Experts.

Figure 7. Preventive measures dictated by Director Gable de Prevencio'n and Promocio'n of Salud

1. - The epidemic outbreak of pneumonia happened in September and October of 1996 in the city of Alcala de Henares was caused by the Legionella bacterium pneumophila SG1, sub-group greater Pontiac, sub-group smaller Knoxville.

2-of the 224 cases including in the outbreak, 92 (41.1%) correspond to confirmed cases, 42 (18.8%) to probable cases and 90 (40.1%) to suspicious cases.

3. - The initial hypothesis of work that oriented the clinical handling of the patients and the medical treatment precociously established, conditioned, to demonstrate the guessed right thing of that one, the favourable general evolution to the treatment in few days, being the rate of lethality caused by this outbreak (4%) more loss than the habitual one in outbreaks of the same nature.

4. - The results of the investigations epidemiologist, microbiological and environmental agree in indicating as origin of the outbreak to one or several cooling towers of water, contaminated with the same stock of Legionella found in the patients. The sanitary deficiencies of the network of potable water supply have been able to play a facilitate able role in the appearance of the outbreak.

5. - At sight of the evolution of the outbreak, the preventive measures, adopted quickly, demonstrated their effectiveness in the control of that one.

6-with the aim to integrate the diverse health actions, directed in his different slopes from the investigation and control of the outbreak, it was necessary to establish precise mechanisms of technical and institutional coordination that worked correctly.

Once personal the outbreaks of legionellosis of Alcala de Henares, the actions of Public Salud was oriented in two fundamental lines: to maintain activated the devices of Vigilancia Epidemiologist before the eventuality of any new case of the disease and of developing to preventive measures forehead to possible new outbreaks
The devices of Vigilancia Epidemiologist have stayed in alert uninterruptedly from the remission of the outbreak to the present time, and will remain at least in alert to the next winter. Its objective is precociously to detect the appearance of possible new cases of legionellosis, with the purpose of taking part as rapidly as possible. Given the characteristics of the past it appears, is logical that the information for the monitoring obtains from the Hospital Prince of Asturias, where were taken care of 96% of the cases of that one. Thus, one has arranged that this Hospital communicates to Service of Public Salud of Area III the following information: Periodic 1.-Notificacio'n (two times per week or exceptionally in case of suspicion) of all pneumonia taken care of (entered or not) with specification of

Name and Last names of the patient.
Date of attention in emergencies. -
Date of birth.
Address.
Diagnosis in Emergencies.
Derivation of the patient.

From this information, and with the purpose of detecting non awaited groups in the time or the space, the incidence and weekly tendency of pneumonia in Alcala de Henares study, comparadolas with the rest of Area III, with observed in the same city years 1993-1996 (epidemic channel of pneumonia) and with the observed thing at the present time and previous years in the geographic zone taken care of by a Hospital of reference in Madrid, like the Gregorio Mara6no'n.
One separately studies the incidence and evolution of the pediatricos cases (where the legionellosis is little frequent) with respect to the cases in adults. Also the place of residence of the cases studies, distribution in a map of the city.
Urgent 2.-Notification of any positive or suggestive microbiological result of recent infection by Legionella known by the Hospital, with the purpose of making the survey pertinent epidemiologist to the case and of investigating the possible source of infection.
The information analyzed by these devices of monitoring epidemiologist allows to affirm that to date the incidence of pneumonias is within the awaited thing and there has been no sprout again of the disease in Alcala de Henares.

Given the possibility, frequently documented in scientific Literature, of appearance of a new outbreak of legionellosis in Alcala de Henares, specially in the months of summer-autumn, when the climatic conditions are more appropriate for it, from the remission of the past it appears has worked intensely in developing a series of tending preventive measures to avoid, in all the possible one, the appearance of that one. The actions made in that sense are the following ones:

1. - Maintenance outside operation of the 6 contaminated cooling towers, during the investigation of the past appears, with Legionella pneumophila SG1, sub-group greater Pontiac, located in the denominated zone of risk (North zone of the municipality) or the next environs, given the implication of the same ones in the origin of the outbreak. These towers could again be put into operation, once fulfilled the requirements established by General Direction of Prevention and Promotion of Salud of Consejeria of Health and Social Services.

2.-Comunicacio'n to the City council of Alcala de Henares of the measures to adopt in relation to the system of potable water supply of its municipality, including: automatic
2.1-Cloracio'n in all the municipal deposits.

2.2.-Mantenimiento of the residual free chlorine levels in all the points of the network in 1.2-1.5 ppm. Continuous and automatic

2.3.-Drenaje of the testable one of the network until a complete one of the same one is made badlyable.

2.4.-Maintenance out of service of the municipal device of water pick up of Smokeable.

2.5.-Estudio in depth of the system of supplying and specifically of relative to pressures of the network and the losses of water, deposits of communities of neighbours, location of points with low or null speeds (testable) and quality of the water in distitos points of the network. All it with the purpose of detecting nonasumibles risks in the network of eliminateable distribution and.

3.-Contract of the services of a qualified company, with the purpose of making a study of evaluation of sanitary risks of the systems or processes of interchange air-water that aerosols generate (evaporative cooling towers, condensers and others) in Alcala de Henares, as well as of the system of potable water supply of this municipality, with specific recommendations (protocols and technical directives of action) for the elimination of the detected risks. The study was made during the February-April months and at the present time already it has been given.

4.-Creation of a Technical Group of Trabajo in Prevention de Legionella in the Community of Madrid, integrated by technicians of General Direction of Prevention and Promotion of Salud and General Direction of Industrial, and Associations of technicians and industrialists related to the maintenance and disinfection of air conditioning facilities and refrigeration. The assignment of this Group has been to review, from the different sectors, enterprise technicians and, the most appropriate boarding (in its slopes technical, operative and legal) for the control of sanitary risks related to the Legionella.

5.-Elaboracio'n of a Resolution of General Director of Prevention and Promotion of Salud, on hygienic conditions that must reunite the evaporative cooling towers, condensers and other devices of interchange air-water, in the municipality of Alcala de Henares. This resolution will be public shortly and contemplates, in relation to the mentioned facilities, the following aspects:
Characteristic techniques. -
Location and height of unloading. -
Maintenance, cleaning and disinfection, before its beginning, whenever been it has stopped a time. -
Treatment and quality of the water. -
Book of incidences. -
Products to use in the cleaning and disinfection. -
enabled Companies to make the maintenance, cleaning and disinfection. -
Sanctions.
Complementarily to the measures adopted by General Direction of Prevention and Promotion of Salud, the City council of Alcala de Henares has approved a regulating Decree of the hygienic conditions of cleaning and of the water tanks of the communities of proprietors.
Once made Resolution public of General Direction of Prevention and Promotion of Salud, and as I complement to the monitoring devices epidemiologist of pneumonia in Alcala de Henares, a device of microbiological monitoring of the water of the facilities with interchange will also be started air-water that generate aerosols, with the purpose of detecting and precociously eliminating possible sanitary risks derived from the contamination of those by Legionella and to evaluate in addition the effectiveness degree of the propose cogoverning measures by Resoluction.

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