Essay: Bacteria, Viruses, and Parasites in Philippines Water

Resorts in the province of Cavite are recognized for its reputable recreational waters. These resorts are intended to provide prospective customers with an atmosphere of amusement, entertainment and relaxation. The most common types are beach resorts, swimming pool, and even lakes and rivers which are designed to accommodate individuals, group of peers and family members (Bago and Linantud 2004). Also recreational waters offer activities that are beneficial and substantial to overall health.

Recreational waters can be contaminated and polluted by bacteria, viruses and protozoan parasites (Bitton 1999), although the recreational water is normally treated physically and chemically using filtration and chlorination to prevent growth and infection of some bacteria (Montano and Abear 2000). However there are was an increasing number of cases of acute gastroenteritis during this summer and one group of microbes leading to their disease are enteric bacteria.

Bacteria such as Escherichia coli and Pseudomonass aeruginosa that are resistant and tolerant to chlorine and were known to cause human misery (Mann 2005). Enteric Bacteria are said to be notorious and dangerous because they cause recreational water illness like acute gastroenteritis, cholera, pneumonia, typoid fever, diarrhea, urinary infection, pneumonia, dermatitis, salmonellosis and otitis external these disease leads to outbreaks (Yoder 2008). Escherichia coli and Pseudomonas aeruginosa are among those bacteria that can thrive in recreational waters and transmitted from swallowing and inhaling contaminated water before the microorganisms can be destroyed by pool water disinfectant (Barwicks et al. 1999). Also their resiliency to grow in some disinfectants like chlorine because they are capsulated bacteria and they possess a versatile metabolic activity, which makes gives them the resistance to a variety of physical conditions (Cappuccino 2005). The presence of these organisms indicates contamination by pathogenic microorganism. Most waterborne diseases are related to pollution of water resources sources and thus pose an unacceptable health risk for swimmers (Schets et al. 2010). Therefore the need to examine water samples in a microbiological water aspect is essential to ensure safety to swimmers.

This study is conducted to support if the selected resorts in Cavite whether chlorinated and non-chlorinate is contaminated with pathogenic microorganism that can lead to potential waterborne diseases.

1.2 Conceptual Framework

The water districts ensure the potability of drinking water by increasing the concentration of Chlorine (Yoder 2008). This potable drinking water were also used in resorts. Monthly sampling of water samples in pools render negative in enteric bacteria. However there is an increasing cases of gastroenteritis for the past year.

The paradigm of the present study is as follows :

Microbial Status in Chlorinated and non- chlorinated waters

from selected resorts

Water samples from resorts (pools, streams, rivers and beaches)

ed

The objective of this study is to gather different water samples obtained from selected resorts and determine the microbial status regardless of its chlorine concentration levels.

1.3 Statement of the Problem

This study will aim to determine the microbial status in selected resorts in Cavite.

To determine specifically the following objectives:

1. What is the microbial status jpresent in chlorinated and non -chlorinated water in selected resorts in Cavite?

2. Is there a significant difference in the total count of enteric bacteria and total bacteria among chlorinated and non-chlorinated waters in selected resorts in Cavite?

3. Is there a correlation between chlorine concentration in the resorts and water samples in lakes, rivers and beaches?

1.4 Scope and Delimitation

The study will determine microbial status in selected resorts in Cavite, Province. Water samples will be taken from these selected resorts and chlorine content concentration will be determined using chlorine test kits. It is not the aim of the study to apply antibacterial agent in enteric bacteria recovered from water analysis.

1.5 Significance of the Study

This study is designed to find out the microbial status on the selected resorts in Cavite, Philippines. This research hopes to benefit the following concerned population:

Resort clientele to be more concerned to the areas that they went to and be prepared since traditional vaccines are not reliable in killing these bacteria because they are risky and are only effective after several years.

Resort Administrators and Maintenance personnel for the enhancement of facilities of the swimming pool to promote the preventive measures against proliferation of microorganism which are recognized to be health risk problems and compliance to the water quality standards.

Academe who may use this as preliminary information for their future research endeavors and information in survival of bacteria in different environmental conditions.

1.6 Definition of Terms

Microbial status – this refers to the bacteria present in chlorinated and non-chlorinated waters from selected resorts.

Prevalence – the number of samples that rendered positive in culture method over the total number of samples.

Enteric Bacteria – these are large group of gram-negative bacteria that are known to produce disease in the alimentary tract. Enteric bacteria that survived in chlorinated waters of resorts.

Resorts – it is considered to be swimming pools, streams, river, lakes and beaches with chlorine.

Microbial Density- The population or the measurement of the growth of the bacteria.

Microbial Plating- This refers technique used to isolate a pure strain from a single species of microorganism plating method that will be performed in laboratory.

Total Bacteria – bacteria other than enteric bacteria.

API Kit- This refers to the biochemical test that will determine the isolated bacteria from water samples.

Chlorination- this is a water purification method to make water safe to humans and a disinfecting agent that prevents the spread the spread of waterborne diseases

Chlorine test kit- This refers to the chemical test that will determine the chlorine concentration level of water.

Chromogenic Media – This refers to the culturing media that will determine the present bacteria in water.

CHAPTER 2

REVIEW LITERATURES

2.1 Conceptual Literatures

Recreational water

Recreational waters can be classified as fresh water swimming pools, whirlpools and naturally occurring fresh marine surface waters. Infectious disease which can be transmitted by recreational water includes skin, eye and ear infections and gastroenteritis. Consequently the level of microorganism in recreational water are important for indexing their health hazard associated with swimming and since the recreation classification includes bathing, swimming etc. any organism transmitted to humans can be regulated. The best indicators in the assessment of the safety of swimming pool water is to become aware of the types of hazard (microbiological, chemical and physical) that can impact a bathing area. Some researchers emphasize that the microbiological quality of swimming pools are best measure by identifying the bacteria present in that recreational water such as fecal coliform and enterococci, while others consider that the disease and symptoms it brought to the bathers rather than fecal contamination (Martin et al. 1995).

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(Montano and Abear 2000) cited that the bacteria suggested as indicators of recreational water quality include a wide variety of pathogenic bacteria and non-pathogenic microorganism such as coliform groups, species of Pseudomonas, Streptococcus, Staphylococcus and in rare case Legionella. The presence of single coliform organism is not a ground for condemning water as a unit for human consumption. It is the relative abundance of these organisms, which is important.

According to Papadopoulou et al. (2007) cited that non-fecal human shedding (e.g. from vomit, mucus, saliva or skin) in the swimming pool is also a potential source of pathogenic organism. Bathers who are already infected can directly contaminate pool waters with pathogen which may affect other bathers, who come in contact with the contaminated water. ‘Opportunistic pathogens’ (mainly bacteria) can also be shed from user and transmitted via contaminated water. Also certain free living aquatic bacteria and amoebae can possibly grow not just in pool waters but also with pool components or facilities or on other wet surfaces within the facility which may cause infections or disease. Therefore swimming pools are often associated with outbreaks or incidents of waterborne infection.

Murdoch(1975) as cited by Amador and Amante (2001) mentioned that disease contracted from water kill some 25million people, most of them children each year, while many millions more are debilitated by waterborne diseases. Fecal contamination of water can introduce a variety pathogens into water waste, including bacteria, viruses, protozoans and parasitic worms. Waterborne related diseases have been recognized by Classes. Class 1, refers to the true waterborne disease contracted by drinking water. Class 2 are diseases associated with lack of personal hygiene which can be reduced by providing adequate amount of water for bathing and washing. To control such diseases, people should be provided with sufficient water of reasonable quality; achieving a high bacteriological quality is a secondary consideration.

Enteric bacteria

A large, heterogenous group in the family Enterobacteriaceae, include several closely related genera of short and spore forming, gram-negative rods, facultative anaerobic, that inhabit or produce disease in the alimentary tract of warm-blooded animal. This family are notorious as causes of urinary tract infection and are recovered from a variety of clinical specimens taken from diseased foci other than in the gastrointestinal tract. The enterobacteria are probably responsible for more human misery than any other group.(Smith 2008)

Escherichia coli

It is a gram negative rod shaped bacterium. It was originally known as bacterium coli. It is widely distributed in the intestine of humans and warm- blooded animals and is the predominant facultative anaerobe in the bowel part of the essential intestinal flora that maintains the physiology of the healthy host. The presence of E.coli is associated with bather-associated illness, but its absence cannot be equated with the lack of risk of illness (Guidelines for Canadian Recreational Water Quality available at http://www.ecy.wa.gov1992).

Pathogenicity performs coliform bacilli usually do no penetrate intestinal wall to produce disease unless (1) the intestinal wall becomes diseased, (2) resistance of the host is lowered, or (3) virulence of the organism is greatly increased. Under one of these conditions of coliforms may pass to abdominal cavity or enter into the bloodstream. Once outside the intestinal canal and in the tissues of the body their virulence is remarkably enhanced. Among the diseases that they cause are pyelonephritis, cystitis, cholecystitis, abscesses, peritonitis, and meningitis. They may play a part in the formation of gallstones and are found in the cores of such stones. In peritonitis complicating intestinal perforation the coliform group is joined by such organisms as streptococci and staphylococci. From any focus of inflammation coliform organism may enter the bloodstream to produce a septicaemia. (Smith 2008)

Shigella

Dysentery caused by the Shiga bacillus (Shigella dysenteriae) is much more severe than that from the other organisms, since this bacillus produces a powerful exotoxin- like substance in addition to an endotoxin. The exotoxin- like substance seems to be liberated by bacterial disintegration, and as a neurotoxin, It acts on the nervous system to paralyze the host. The endotoxin irritates the intestinal canal.

The dysentery bacilli are gram negative, nonsporebearing rods that grow on all ordinary media at temperatures from 10° to 42° C. but best at 37° C they are aerobic and facultative anaerobic. Unlike most other members most other member of the enteric group, they are non-motile.

In terms of pathogenicity dysentery is a human disease and natural infections of the lower animals do not occur. The incubation period is 1 to 7 days. Epidemic dysentery is primarily an intestinal infection. Unlike typhoid bacilli, the organisms do no invade the bloodstream and are seldom if ever found in the internal organs or excreted in the urine. They are excreted in the feces. Compared to that for other enteric pathogen, the number of ingested shigellas for infection is small, only 10 to 100. (Smith 2008)

Salmonella

Among the large number of pathogenic microorganisms causing foodborne disease, Salmonella plays an important role. An analysis of Salmonella surveillance data from the World Health Organization (WHO) showed that the reported number of cases increased in 22 out of 49 countries examined. Although the reason for the global increase is not yet clear, investigations in individual countries suggest that it is related to consumption of eggs and poultry that harbour the organism. Besides control measures there is a need for rapid and sensitive methods for the detection of Salmonella (Beumer et. al, 1991). Salmonella is a ubiquitous enteric pathogen with a worldwide distribution that comprises large number of serovars characterized by different host specificity and distribution. This microorganism is one of the leading causes of intestinal illness through the world as well as the etiological agent of more severe systemic diseases such as typhoid and paratyphoid fever.

Zoonotic salmonellae are commonly described as foodborne pathogens however; drinking water as well as natural waters is known to be an important source for the transmission of these enteric microorganisms. Salmonella, just like other enteric bacteria, is spread by the fecal-oral route of contamination. This microorganism can enter the aquatic environment directly with feces of infected humans or animals or indirectly, e.g., via sewage discharge or agricultural land run off.

Overall Salmonella spp. and subspecies can be found in a large variety of vertebrates. Beside humans, animal sources of Salmonella include pets, farm animals and wild animals; calves, poultry, pigs, sheep as well as wild bird (pigeon) and reptiles can all be reservoirs of Salmonella. Plants, insects and algae were also found capable of harboring Salmonella and might be implicated in the transmission of this enteric pathogen. Taxonomically the genus Salmonella comprises two species namely S. bongori and S. enterica. The species S. enterica is further differentiated in to six subspecies (enterica, salamae, arizonae, diarizonae, indica and houtenae) among which the S. enterica subspecies enterica is mainly associated to human and other warm blooded vertebrates. Enteric fevers, typhoid and paratyphoid fever are severe, contagious systemic diseases caused by the infection of the serovars typhi and Paratyphi. Differently from other Salmonella serovars, typhi and Paratyphi are host adapted and can only infect humans; stools of infected persons are therefore the original source of contaminations for these pathogens.

Water contaminated with feces of human cases and carriers is one of the main vehicles of typhoid fever infections. Literature data related to water-borne salmonellae in developing countries relate mostly the typhoid Salmonella serovars. In the less industrialized area of the world, in particular in the Indian subcontinent and South East Asia, typhoid and paratyphoid fevers occur both in epidemic and endemic form, and remain a major public health problem. The burden of typhoid fever worldwide is further compounded by the spread of multiple drug resistant S. typhi.

Most of the recent publications on typhoid and paratyphoid fever water-borne infections in developing countries are from the Asian continent. Differently from typhoidal Salmonella strains, non-typhoidal salmonellae, the ubiquitous subtypes found in a number of animal species, are more frequently associated to foodborne than to water-borne transmission. These zoonotic Salmonella serovars tend to cause acute but usually self-limiting gastroenteritis (Levantesi et al, 2011).

According to (Smith 2008)The pathogenicity of salmonella is called salmonellosis, the major site of which the lining of the intestinal tract. Because of their toxic properties every known strain of salmonella can cause anyone three types of salmonellosis: (1) acute gastroenteritis of the food type infection.(2) septicemia or acute sepsis with localized complications similar to pyogenic infections, and (3) enteric fever such as typhoid or paratyphoid fevers.

Salmonella typhi

A short motile nonencapsulated bacillus, S.typhi grows luxuriantly on all ordinary media. It grows best under aerobic conditions bit may grow anaerobically. The temperature range growth is from 4° to 40°C., the optimum, 37°C. typhoid bacilli can survive outside the body, living about 1 week in sewage contaminated water and not only living but multiplying in milk. They may be viable in fecal matter for 1 or 2 months. They are pathogenic because of their endotoxins.

Their pathogenicity causes typhoid fever is an acute infectious disease with continuous fever, skin eruptions, bowel disturbances, and profound toxemia. Except in the first few days, leukopenia is always present in uncomplicated cases, probably because typhoid bacilli depress the bone marrow, where normal production of white blood cells occurs. Leukocytosis in the course of the disease signals complication. (Smith 2008)

2.2 Related Studies

According to Brown (2009), gram-negative intestinal pathogens have a diverse population of bacteria of which two of the enteric intestinal pathogens that are of prime medical concern are the salmonella and shigella. The salmonella and shigella are both pathogenic bacteria that cause typhoid fever and human dysentery, respectively. Since the gram-negative intestinal pathogens has a such diverse population it has many genera of species like the Escherichia, Proteus, Enterobacter, Pseudomonas, and Clostridium that exists on large numbers, hence it is necessary to use media that are differential and selective to favor the growth of the pathogens since all of the species can be divided into lactose fermenting and non-lactose fermenting bacteria.

Hiriart et al. (2001) worked on the Helicobacter pylori and Other Enteric Bacteria in Freshwater Environments in Mexico City. They observed that all samples analyzed showed the presence of enteric bacteria with or without the presence of H. pylori, indicating that water from these sources is a potential health risk for gastrointestinal diseases. The major positivity of H. pylori coincides with the major positivity of indicator and other enteric bacteria, which are both associated with contaminated water.

In another study Marion et al. (2010) worked on the association gastrointestinal illness and recreational water exposure at an inland U.S beach. Relationships between water quality indicators and reported adverse health outcomes among users of a beach at an inland U.S lake was observed to be a significant risk factor for GI illness.

.

Papadopoulo et al.(2008) worked on the microbial quality of indoor and outdoor swimming pools in greece. They found out that three indoor swimming pools and two outdoor swimming are present with bacteria, protozoa and fungi Such as Multi-resistant Pseudomonas alcaligenes, Leuconostoc, and staphyloccus aureus( isolated from teaching pool), Staphylococcus werneri. Chryseobacterium indologenes and Ochrobactrum anthropic (isolated from completion pools) Pseudomonas aeruginosa, P. fluorescens, Aeromonas hydrophila, Enterbacter cloacae, Klebsiella pneumonia and S. aureus (isolated from the hydrotherapy pool and A. hydrophilla (isolated from the hotel pool) were related to water outbreaks.

Schets et al. (2010) worked on the exposure assessments for swimmers in bathing waters and swimming pools. they found out that the swallowed volume or water appears different for men, women, and children, but also in fresh water, seawater and swimming pools also the frequency and duration of swimming do also differ for men, women, and children and in different water types, and provide a basis for the identification of high risk population under specific circumstances, e.g. due to their extended water contact and frequent head submersions, children may be more prone to contract otitis external due to Pseudomonas aeruginosa infections.

Certainly a waterborne infection depends on the total bacterial counts, the immune status of the subjects, and polluted waters. The results of the past studies demonstrate the variability of the recreational water quality and the need for continuous monitoring.

Chapter 3

METHODOLOGY

Research Design

This study will use descriptive study design that involves in the identification of enteric bacteria in selected resorts in the Cavite province. There will be 20 sampling sites, 10 from swimming pools, 5 from rivers or lakes and 5 from beaches. In every sampling site there will be a total of 1 sample that will be gathered and it will be replicated into three and a total of 60 sterilized bottles with cover will be used for the 4-month period of experiment that will be done during the summer season and the rainy season.

Research Setting

The entire study will be conducted for 12 weeks. The identification of total bacteria and enteric bacteria will be done in Biology Research Laboratory of DLSU-D.

Research Procedure

Water Sample Collection (MicroMed Environmental, 2010)

Sterilized 300ml wide-mouthed glass will be used in the collection of samples. Water samples will be obtained from recreational waters. The sterile containers will be plunge into the water surface until 1 foot below. Then open the bottle towards the direction of the current to allow the container to fill. Afterwards, it will be immediately sealed tightly and placed on a cooler to maintain the temperature. The samples will be obtained during the months of april and june of 2012. The chlorine concentration will also be measured using Hach Test Kit for chlorine.

Chromogenic Media for Bacteria

Undiluted samples will be used in the determination of total bacteria. Briefly one milliliter of sample will be spread plated onto Plate Count Agar. The plates will be incubated at 37°C for 24 hours. Colonies that will grow will be converted into colony forming units and will be correlated to chlorine concentration and compared to enteric bacteria.

For the detection of enteric bacteria the samples will be enriched in buffered peptone water for 24 hours. After 24 hours the enriched samples will be spread plated onto Salmonella-Shigella Agar and Eosin Methylene Blue Agar. Colonies resembling to enteric bacteria will be purified and confirmed using API 20E kit.

Determination of the Microbial Count (BioMérieux, 2002)

Preparation of incubation box and inoculum will be done for the strip. In the inoculation of the strip, filling both tube and cupule of tests CIT, VP and GEL with bacterial suspension as for the remaining tests fill only the tube and not the cupule. In creating anaerobiosis ADH, LDC, ODC, H2S and URE should be overlay with mineral oil. The incubation box will be incubated for 37°C for 24 hours. Certain color reactions will happen for the indication of positive or negative result.

Data Gathering

Colonies in the EMBA and PCA will be characterized using colonial characterization which includes size, form, margin, elevation, consistency, surface and pigmentation (Tabo, 2005). Biochemical test include ONPG, ADH, LDC, ODC, CIT, H2S, URE, TDA, IND, VP, GEL, GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA, OX. The chlorine concentration will be measured in 0-600 mg/L.

Statistical Treatment

To determine the correlation between chlorine concentration and total bacteria and enteric bacteria, a simple correlation will be used. All statistical analysis will be conducted in STATA 9.0 with 0.05 as level of significance.

APPENDIX A

GANTT CHART

APPENDIX B

BUDGET PROPOSAL

Item

Volume/Mass

Estimated Price (PhP)

Quantity

Expense (PhP)

EQUIPMENTS AND KITS

Biomerieux Inc Biomerieux API 20E KIT 100g Pack of 100 20160

–

13000.00

1

13000.00

Hach’s Chlorine Test Strips, 0-600mg/L Pack of 2890200

–

876.31

1

876.31

AGARS

Salmonella-Shigella Agar

50 g

500.00

1

500.00

Eosin Methylene Blue Agar

50 g

500.00

1

500.00

Plate Count Agar

–

500.00

1

500.00

TOTAL

15376.31

APPENDIX C

LETTER TO THE HOSPITAL

March 13, 2012

Ms. Teresita E. Guevarra

Medical Records Head

De La Salle University Medical Center

Dear Ms. Guevarra:

Greetings in the name of St. John Baptist De La Salle!

We are writing to ask permission from you in getting information that we will need for our thesis defense on the upcoming December 2012. We are Human Biology major students from De La Salle University-Dasmariñas and we are going to conduct a study regarding the possible prevalence of enteric bacteria in selected resorts in Dasmariñas, Cavite. Regarding this, we would like to request for the following information:

Reported cases of salmonellosis and acute gastroenteritis in this hospital for the last two years (2010 and 2011)

We are hoping for your positive response towards our request. If ever the information we need will not be available today, you may contact us at 09164745448 and 09272546946. Thank you very much for your time.

Sincerely,

Ron Matthew A. Flores

John Paul A. Flores

Noted by:

____________________ _____________________

Mrs. Hazel Ann L. Tabo Dr. Carmelita C. Cervillon

BSD Faculty, DLSU-D (Thesis Adviser) College Dean, DLSU-D

_____________________

Ms Cherry Z. Cuevas, MS

BSD Chair, DLSU-D

APPENDIX D

COLOR REACTION

TESTS

– RESULTS

(negative)

+ RESULTS

(positive)

ONPG

colorless

yellow

ADH

Yellow

red/orange

LDC

Yellow

red/orange

ODC

Yellow

red/orange

CIT

pale green/yellow

blue-green/blue

H2S

colorless/gray

black deposit

URE

Yellow

red/orange

TDA

Yellow

brown-red

IND

Yellow

red (2 min.)

VP

colorless

pink/red (10 min.)

GEL

no diffusion of black

black diffuse

GLU

blue/blue-green

yellow

MAN

blue/blue-green

yellow

INO

blue/blue-green

yellow

SOR

blue/blue-green

yellow

RHA

blue/blue-green

yellow

SAC

blue/blue-green

yellow

MEL

blue/blue-green

yellow

AMY

blue/blue-green

yellow

ARA

blue/blue-green

yellow

OX

colorless/yellow

violet

LITERATURE CITED

Amador RM, Amante PP. Detection and isolation of coliform bacteria in Laguna de Bay Brgy. Landayan San Pedro Laguna; 2001. p.67.

Bago CEM, Linantud JF, Ortiz MP. Stability and Profitability of Resort Business in Dasmarinas, Cavite. 2004. P.1-2-ix-29.

Barwicks RS., Levy DA., Craun GF., Beach MJ., Calderon RL. 2000.

Surveillance for water borne-Disease Outbreaks-united-states ,1997-1998 CDC

Brown, A. E. 2005. Benson’s Microbiological Applications 9th Edition, McGraw Hill, New York.

Beumer, R.R., et al., 1991. Enzyme-linked immunoassays for the detection of Salmonella spp.: a comparison with other methods, Elsevier Science Publisher, B.V. 0168-1605/91

Carteciano JA., 2004. Four Emerging Bacteria: So Tiny, So deadly. National

Research Council of the Philippines.

Hammer Sr. M, Hammer Jr. M. Water and waste water technology. New Jersey; 2004.p.140

Levantesi, C., et al., 2011.Salmonella in surface and drinking water: Occurrence and water-mediated transmission, Food Research International, doi:10.1016/j.foodres.2011.06.037;

Mann, D. Beware of Recreational Water Illnesses, WebMD. [Internet]. 2005 [cited 2011 December 28].

Available from HYPERLINK “http://www.webmd.com/fitness-exercise/features/beware-of-recreational-water-illnesses”

Marion, J., et al., 2010.Association of Gastrointestinal illness and recreational water exposure at inland U.S beach, water research international;

Martin, M., et al., 1995.Assessment of microbiology quality for swimming pools in South America.

MicroMed Environmental, Inc. [Internet]. 2010 [cited 2012 March 25]. Available from HYPERLINK “http://www.igmicromed.com/docs.html”

Montano JM, Abear R. 2000.Detection of Pseudomonas aeruginosa in relation to microbial population of selected swimming pools in dasmarinas cavite. De la Salle University Dasmarinas. p.52.

Schets F., et al., Exposure Assessment of swimmers in bathing water and swimming pools, water research. 2010.

Tabo, Norbel A. 2005. Laboratory Manual in Microbiology, Rex Bookstore Inc, Manila. p. 63-67

Yoder JS., Hlavasa MC., Craun GF., Hill V., Roberts V., Yu PA., Hicks LA., Alexander NT., Calderon RL., Roy SL., and Beach MJ.2008. Surveillance for waterborne disease and outbreaks associated with recreational water use and other aquatic facility- associated health events-united states 2005-2006- CDC.