Title: Epidemiological and virological investigation of a Norovirus outbreak in a resort in Puglia, Italy

Transcription

1 Author's response to reviews Title: Epidemiological and virological investigation of a Norovirus outbreak in a resort in Puglia, Italy Authors: Caterina Rizzo (caterina.rizzo@iss.it) Ilaria Di Bartolo (ilaria.dibartolo@iss.it) Marilina Santantonio (marilinasantantonio@tiscali.it) Maria F Coscia (mf.coscia@igiene-seconda.uniba.it) Rosa Monno (r.monno@igiene-seconda.uniba.it) Danila De Vito (d.devito@igiene-seconda.uniba.it) Franco M Ruggeri (franco.ruggeri@iss.it) Giovanni Rizzo (g.rizzo@igiene-seconda.uniba.it) Version: 2 Date: 5 September 2007 Author's response to reviews: see over

2 Dr. Erik Alexandersson, PhD Assistant Editor BMC-series Journals BioMed Central RE: revision of MS: Epidemiological and virological investigation of a Norovirus outbreak in a resort in Puglia, Italy Rome, XX/07/2007 Dear Dr Alexandersson, we enclose a revised manuscript MS: entitled " Epidemiological and virological investigation of a Norovirus outbreak in Puglia, Italy" by Caterina Rizzo, Ilaria Di Bartolo, Marilina Santantonio, Maria F Coscia, Rosa Monno, Danila De Vito, Franco M Ruggeri and Giovanni Rizzo. The paper has been revised according to the suggestions of the three referees, as detailed in the enclosed notes. We hope that the revised manuscript is clearer thanks to these reviews and is now suitable for publication in BMC- Infectious Disease. Sincerely, Caterina Rizzo, MD, for the authors.

3 Reply to the reviewers comments MS: Reviewer #1: Reviewer's report Epidemiological and virological investigation of a Norovirus outbreak Title: in Puglia, Italy Version: 1 Date: 15 June 2007 Reviewer: Alicia Sanchez-Fauquier Reviewer's report: General Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) The authors refer to the occurrence of a large outbreak of NoV gastroenteritis occurred in the region of Puglia however they don t study any case outside the resort. That is why it should be changed by A large outbreak involving 400 people in a Resort of Puglia region, or otherwise to explain how many people out of this resort were involved and tested. It would be interesting to know how many cases occurred outside the resort as well as if the water source was the same in both cases. Response: This is an interesting point; we add a comment in the discussion ( During the same period outside the resort no other NoV outbreaks were notified in the same area. Moreover, a recent study in a close area (Taranto province, see Figure 1), has shown that during 2005 the number of patients with gastroenteritis seeking hospital care was lower compared to 2006 (26) ). Unfortunately, there is no information on cases outside the resort because Norovirus disease is not included in the specific diseases that are subject to mandatory notification, as part of the mandatory notification system for infectious diseases (SIMI), coordinated at the national level by the Ministry of Health. SIMI categorizes notifiable diseases into 5 classes, according to severity. Class IV diseases include any disease that occurs as an outbreak. No other NoV outbreaks were notified in the same period, in the same area. For these reasons we were able to detect the outbreak in the resort. We rephrased in the revised version ( In the present study, we describe the occurrence of a large outbreak involving 400 people in a Resort of Puglia region in July 2005 ). Despite of Elisa is described under methods of detection they don t comment or in results or in discussion how many cases were detected by using this technique. Response: We agree with the reviewer, we add a paragraph in the discussion section regarding the comparison of both techniques ( We used both NoV-specific ELISA and RT-PCR to test 20 faecal samples collected during the outbreak investigation. Using ELISA test, 2 (5%) of the 20 samples were positive for GII antigens. The RT- PCR test detected NoV RNA in 18 (90%) samples, including the two positive by ELISA. These results fit with previous findings that ELISA assays may not show a similar sensitivity as RT-PCR methods (29), although ELISA might represent a simple and rapid diagnostic test for timely investigatios of NoV outbreaks in laboratories with low capacities. ) However, in the first paragraph of the Laboratory investigation result section it was already indicated that Two stool samples obtained from 20 patients, resulted positive by search for NoV-specific ELISA. Figure 1 should be deleted due to the information that 18 samples were positive by using RT-PCR+ would be enough. Response: We agree with the reviewer and we deleted this figure. How many samples were performed with each technique? Is it possible to compare the results of both techniques? Response: We complied with these comments; see reply above. Were there other viral agents detected? Response: This is an interesting point, based on clinical symptoms and age of patients we firstly performed NoV-ELISA test, then confirmed results by RT-PCR. Since the characteristics of the outbreak also fit well with a NoV etiology, we excluded other viral agents from our research. In figure 2 the first case appears on the tenth day, two days later two more cases and since the fourteenth day the outbreak bursts. This fact could suggest the norovirus transmission person to person, this possibility has not been considered.

4 Response: We agree with the reviewer that the epidemic curve was confounding, and according also to other reviewers comments, we deleted this figure. We also commented a possible contribution of person-to-person transmission to expansion of outbreak in the Discussion section ( In addition to a point source of infection, a person to person transmission of Nov during the outbreak is also likely to have occurred as supported in particular by both the occurrence of few isolated cases in the first days of the outbreak before the peak four days late, and the occurrence secondary cases within families. ) The only reason why they concluded the infection origin is the water is for the presence of faecal bacteria. Would be possible to know the data of water off the resort during the period of the study? Response: This is an interesting point; unfortunately, no microbiological data concerning the water outside the resort were available at the Local Agency for Environment. Another question to be answered is their consideration about important risks factors as the ingestions of mussels and ice cream anyhow they conclude that the cause of the norovirus infection is the water without testing it. Response: We agree with the reviewer. We commented on this point in the discussion section. In this paper would be advisable to change and clear up important questions as the epidemic curve. It seems difficult to get epidemiological global conclusions studying only a week of the outbreak. (41 cases grouped in 9 days) instead in 3 weeks as it was written under discussion. Response: We commented on this. See Response above. Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) Discretionary Revisions (which the author can choose to ignore) What next?: Unable to decide on acceptance or rejection until the authors have responded to the major compulsory revisions Level of interest: An article of limited interest Quality of written English: Not suitable for publication unless extensively edited Statistical review: Yes, but I do not feel adequately qualified to assess the statistics.

5 Reviewer #2: Reviewer's report Title: Epidemiological and virological investigation of a Norovirus outbreak in Puglia, Italy Version: 1 Date: 26 June 2007 Reviewer: Yvonne T Van Duynhoven Reviewer's report: General In general this paper on the third norovirus outbreak in the Southern region of Italy is not a very innovative paper. It is just another outbreak, with a already known source(s). However, the epidemiological component of the investigation is well-done, analysed and described. But, the food- and environmental study components are suboptimal. Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) - The conclusions about the water being the source in the abstract really does not fit with the data presented in the results. The mussels and ice are the ones you expect to see there. Only in the discussion some arguments are provided for the water source. To my impression, the mussels are just as likely, as they are a well-known vehicle for norovirus, than the water source. I believe the abstract conclusion and the discussion should be adapted accordingly. Response: We agree with the reviewer. We modify the abstract conclusion ( The most likely hypothesis supported by the findings of the epidemiological investigation was that illness was associated with raw mussels and ice. These hypothesis could not be confirmed by specific microbiologic testing for NoV in food or ice. ) and the discussion. - page 8 states that food samples were not collected. It is not clear why not, as that would be a prerequisite in such an outbreak investigation. Related to that: in the discussion it was stated that no left overs were available for testing, but the end of the 1st paragraph of the discussion says that no further cases developed when a.o. serving raw mussels was stopped and disposal of previously stored ice!, was implemented. Please clarify. Response: We were unclear. We clarified this point in the different sections, especially at the end of the first paragraph in the discussion section ( No further cases were reported after July 29, when specific public-health measures were implemented (such as: drinking only bottled water, super-chlorination of the resort tank, stopping the serving of raw mussels, and disposal of previously stored ice). ). - it is unclear form the methods section how the case finding was done, by which the authors arrive at the 400 cases among hotel guests. Response: We clarify this point in the methods section ( Case finding was done by a door-to-door search within the Hotel ). - could the authors elaborate on whether the water could have been contaminated by the ill guests, as sampling was done at the end of the outbreak, and NoV is famous for their stability in the environment. How was tap water sampled? (sterile sampling?) Response: We agree with the reviewer. We added a comment in the discussion section, and we also add a specific comment in the method section on how tap water was sampled ( water samples were consecutively collected from various sites, in sterile condition, inside the resort and examined by the Local Agency for Environment. ) - What kind of ice was incriminated by the epi investigation? Ice cubes made of tap water or ice provided by a supermarket? The conclusion indirectly suggests a link between ice and contaminated water. This was not clear from the previous parts of the paper. Response: Sorry, we were unclear. The ice cubes were made of tap water. We clarified this point in the different sections of the paper. - Conclusions, page 13: control measures had no effect: Which ones were meant, as on page 11 some measures are mentioned which seemed to have stopped the outbreak? So, there seems to be some discrepancy in the description there. Response: We agree with the reviewer. We clarify this point in the conclusions section ( Before July 23, no control measures to limit the spread of the infection were implemented, probably because the local health unit did not address the point source and failed to prevent person-to-person transmission. )

6 Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) - The last sentence of results in the abstract NoV was miss-spelled as NOV. - The last sentence of the conclusion of the abstract is not a good English formulation (too long sentence anyway. Better to cut them in two small sentences). - page 3, references by accident include a comma. -page 5,..'were matched for age and gender' can be excluded, as this was already clarified in the previous sentence. -page 6, lab investigations, 1st sentence: 'the' should be added before case definition. -most part should be majority, In the same sentence at the end 'than the controls' should be added. - discussion, page 11, 'a' should be added before few days. - page 12, showed before ref (12) should be shown. Same sentence, part should be role. - page 12 sentence before ref 31,32, strain should be plural: strains. Discretionary Revisions (which the author can choose to ignore) - In the background of page 3, reference 7 and 8 are really out-dated. There is numerous more recent literature, better covering the statement. - Also, only person-to-person transmission is mentioned, while both transmission through contaminated food and water often apply as well. Response: We agree with the reviewer, in fact in the background is indicated clearly the route of transmission of Norovirus infections: ( Transmission occurs person-to person by faecal-oral route or aerosol formation. ). The faecal-oral route is possible by food or water consumption. - In the 3rd paragraph of the background, last sentence, it would be good to repeat that you are discussing the Italian situation, as this does not apply world-wide. Readers might otherwise easily misinterpret this part. ( Therefore the burden of NoV infection in Italy is presently unknown, and little information is available about circulating viral strains. ) - Page 4, last paragraph, better to say that the paper describes the investigation instead of the occurrence of the outbreak.

7 - How was the onset period for the cases (2 to 25 July) chosen? Response: The onset period was 2-19 July. Sorry this was due to a typing error. We define the onset period starting from the first day in which the cases were reported to the Hotel staff (personnel communication from the local heath unit) and the epidemiological investigation day July Did the questionnaire ask for water consumption? If yes, what were the results of the univariate analyses for these items? Response: Yes, there was a question regarding the water consumption. The univariate analysis revealed a Risk Factors of 0.9 with p-value of In the manuscript, only Risk Factors with p-value<0.20 were reported, for this reason we excluded the water consumption. - The last paragraph of the section outbreak investigation on page 9 can be deleted, as it is not of value. Response: According to reviewer suggestion, we deleted the paragraph. - Discussion, page 11: The sentence The delay in... would better be cut in two shorter sentences. - Discussion, 2nd paragraph: do not repeat exactly the results. The first three sentences can easily be deleted. What next?: Reject because too small an advance to publish Level of interest: An article of insufficient interest to warrant publication in a scientific/medical journal Quality of written English: Needs some language corrections before being published Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interests

8 Reviewer #3: Reviewer's report: Title:Epidemiological and virological investigation of a Norovirus outbreak in Puglia, Italy Version: 1 Date: 11 June 2007 Reviewer: Preben Aavitsland Reviewer's report: General This in an interesting investigation of an outbreak of gastroenteritis in an Italian hotel. The causative agent was norovirus. Because of the many potential transmission routes of this pathogen, and its ability to cause long drawn epidemics in closed communities as hotels and cruise ships, it is very difficult to pinpoint what started an outbreak. This is also the case here. The authors try to make the cases that tap water caused the outbreak, but the evidence is mostly circumstantial. It seems that they could use their current data to argue even better for the primary role of drinking water. Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) Page 5. Were the cases guests and employees while the controls were residents only? Is this a potential cause of bias? Response: At the beginning of the epidemiological investigation we collected questionnaires also from the 28 staff members of the hotel, and they were initially included in the analysis. Unfortunately, data collected from the staff members of the hotel were not complete and answers related to food and water consumption were mostly missing. For this reason, we eventually decided to exclude the staff members from the analysis to be reported in the manuscript. Nonetheless, while editing the manuscript we failed to delete the results concerning the staff members in some parts of the text. We edited the manuscript deleting all the ambiguous parts. Page 5. How were the cases selected? And the controls? Response: Please see previous response #3 to Reviewer #2 on case finding. Page 11. How could you use the questionnaires of cases and controls to calculate an attack rate? Response: Apologies. We made an editing error (see, the above response #1) and forgot to delete that paragraph. We deleted that paragraph in the discussion section. Page 12. I think you need to explain more thoroughly why you think drinking water was the primary source. At the time you did your investigation, the infection had been present in the hotel for a couple of weeks, and many of the staff were infected, and also there may have been environmental contamination. In addition, one would assume that person to person-transmission played some part. Some supporting information may be: Were the ice cubes made from tap water or bottled water? Had the raw mussles been in contact with the tap water? Did you ask about drinking tap water? Or showering in the hotel? (To pose a different hypothesis: Could the outbreak have started by contamination of food by an infected kitchen employee and then be propagated by food and ice cubes plus person to person transmission?) Response: We agree with the reviewer on the importance of better pointing out the drinking water as a primary source. We have addressed these points in previous responses to the other reviewers,we. focused better on the point that the ice cubes were made of tap water. Regarding the mussles been in contact with tap water, we had two different version: 1) food-handlers, at the beginning, excluded this eventuality whereas, 2) the local health unit did not exclude this practice. For this reason, we had excluded this comment from our discussion. Please see additional specific comment in the Discussion section ( In addition to a point source of infection, a person to person transmission of Nov during the outbreak is also likely to have occurred as supported in particular by both the occurrence of few isolated cases in the first days of the outbreak before the peak four days late, and the occurrence of secondary cases within families. More simultaneous transmission routes have indeed been reported previously shown (12) ). Page 12. What happened to symptomatic staff? Were they excluded from work? Response: Please see previous response #1 Figure 1. It seems a bit unusual to draw an epidemic curve based on only a selected group of patients. Usually, we draw the epidemic curve based on all cases. Did you investigate how many of the residents who had the disease? And were these 41 representative of all? I am not convinced that this epidemic curve indicates a common source. Response: We agree with the reviewer, and according also with other reviewers comments, we deleted this confusing figure.

9 Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) Page 3. Please mention that the three outbreaks (ref 12, 18, 19) were in Italy. Page 9. No need to specify that cases met the case definition. Discretionary Revisions (which the author can choose to ignore) Page 7-8. The description of the virological methods is very long. Is this a standard method that has been described before so that you can refer to another publication and shorten the description here? Response: The description of RT PCR methods has been substantially reduced, as requested by the reviewer. What next?: Unable to decide on acceptance or rejection until the authors have responded to the major compulsory revisions Level of interest: An article of limited interest Quality of written English: Needs some language corrections before being published Statistical review: No, the manuscript does not need to be seen by a statistician. Declaration of competing interests: I declare that I have no competing interests.