September

Dedication

Dedicated to my beloved parents

Certificate

Dr. S. JEYACHANDRAN, M.Sc., M.Phil., Ph.D. Residence Research Adviser and Associate Professor, No. 13, Gowtham Nagar A.V. V. M. Sri Pushpam College (Autonomous), Nagai road, Poondi – 613 503, Thanjavur District, Thanjavur – 613 001 Tamil Nadu, India Tamil Nadu, India e-mail: [email protected]

CERTIFICATE This is to certify that the thesis entitled “ Studies on the Atmospheric Microflora and its Allergenicity in Chennai, Tamil Nadu, South India” submitted to Bharathidasan University, Tiruchirappalli – 620024, for the award of the degree of Doctor of Philosophy in Microbiology, embodies the result of the bonafied research work carried out by Mrs. S. Vijayalakshmi, under my guidance and supervision during the study period at the Department of Botany and Microbiology, A. V. V. M. Sri Pushpam College (Autonomous), Poondi – 613 503, Thanjavur District, Tamil Nadu, India.

I further certify that no part of this thesis has been submitted anywhere else for the award of any degree, diploma, associateship, fellowship or other similar titles to any candidate.

Dr. S. JEYACHANDRAN Research Adviser

Declaration

DECLARATION

I do hereby declare that this work has been originally carried out by me under the supervision of Dr. S. JEYACHANDRAN, Associate Professor, P. G. and Research

Department of Botany and Microbiology, A. V. V. M. Sri Pushpam College (Autonomous),

Poondi – 613503, Thanjavur District, Tamil Nadu, and this work has not been submitted elsewhere for any other degree.

September, 2010. Poondi – 613503 (S.VIJAYALAKSHMI)

Acknowledgement

ACKNOWLEDGEMENT

I owe a great many thanks to a great many people who helped and supported me during the writing of this thesis.

Words are inadequate in offering my deep sense of gratitude to my principled research guide, Dr. S. Jeyachandran, Associate Professor, Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College, Poondi, Thanjavur, for his precious guidance. With his enthusiasm, his inspiration, and his great efforts to explain things clearly and simply, he helped throughout my thesis – research period, with lots of encouragement, sound advice, and good innovation.

I express a great privilege to thank Sri K. Thulasiah Vandayar, Secretary and Correspondent, and Dr. S. Chinnaiyan, Principal, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur, for their kind support and concern for providing the required facilities to carry out my research work. My sincere thanks to Dr. A. Thayumanavan, Dean, Faculty of Science, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur for his moral support and encouragement.

I sincerely extend my thanks and gratefulness to my doctoral committee member Dr. S. Kulothungan, Associate Professor, Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur for his thoughtful suggestions and support during my research.

I am indebted and owe my due respects to Dr. R. Chandrasekaran, Co-ordinator and Associate Professor, Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur, for his gratitude and constant support.

My heartfelt thanks to, Dr. C. Manoharan, Associate Professor, Department of Botany and Microbiology, A.V.V.M. Sri Pushpam, Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur, who had been a source of inspiration for his timely guidance and suggestions in the conduct of my research work at all the crucial stages of my study.

I also express my cordial thanks to, Dr. T. Selvaraj, Former Head, Dr. A. Panneerselvam, Dr. T. Kumar, Mr. C. Chandran, Dr. V. Ambikapathy,

Dr. S.Vijayakumar, Faculty members, Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur for their kind hearted support.

I have great pleasure in expressing my heartfelt thanks to Dr. S. Gurumani, Former Head, Department of Zoology and Microbiology, Pachaiayappa’s College, Chennai for his timely support and keen interest in delivering precise statistical data in every phase of the work.

I express my sincere thanks to Dr. S. Narasimhan, Principal, D. G. Vaishnav College, Chennai for his vital support and encouragement throughout my tenure of my research study. I record my gratefulness and courteous thanks to Mrs. P. Vidhya, Head, Department of Microbiology, D. G. Vaishnav College, Chennai for initiating me to sustain a consistency on my research. I would like to express my gratitude to Mr. Prabhakaran, for his influential help to carry out the research.

I am indebted to my college colleagues Mrs. Radhika Jevanand and Mr. Jevanand for providing a stimulating and fun filled environment for helping me to get through the difficult times. I am also grateful to Mrs. S. Jagadeeswari, Mrs. Kirthiga Suresh and Mrs. Nisy for all the invaluable help, emotional support and caring they provided.

My sincere thanks to Dr. R. Rajendran, Reader, Department of Microbiology, PSG College of Arts and Science, Coimbatore for his thought provoking suggestions at all the stages of my research work.

I also record my courteous thanks to my professors Dr. N. Kannan and Dr. R. Selvakumaran, Former heads, Dr. S. Bakthavatsalam, former faculty, Dr. K. Radha, Senior Lecturer, Dr. A. Michael, Head, Department of Microbiology, PSG College of Arts and Science, Coimbatore, for their encouragement.

I extend my great privilege to my undergraduate lecturer, Dr. Aruna Devaraj, Dr. N. Arunagirinathan, Mr. P. Sivamani and Mrs. Geethavani for their help and wishes for the successful completion of this project. I also wish to record my heartfelt thanks to my nephew Mr. V. Padmanabhan for his timely support.

My lovable and affectionate thanks are due to my parents, Sri. T. S. Srinivasan and Smt. S. Lakshmi, husband Mr. V. Ramesh, daughter R. Jayashree, family members and friends for their patience and wishes for the successful completion of this research.

S. Vijayalakshmi

Contents

CONTENTS

Page No

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 7

2.1. Bioaerosols 7 2.2. Air borne 10 2.3. Sampling methods 18 2.4. Culture methods 22 2.5. surveys: The professional approach 24 2.6. Efficacy data and labelling requirements of Air sanitizers 26

3. MATERIALS AND METHODS 27

3.1. Sampling site 27 3.2. Survey protocol 27 3.3. Schedule of air sampling 27 3.4. Air sampling methods 28 3.5. Identification of 30 3.6. Identification of fungi 33 3.7. Presentation of data 3.8. Recording of meteorological parameters 34 3.9. Statistical analyses 35 3.10. Immunological analysis 35 3.11. Air sanitation 39

4. RESULTS 41

4.1. Microbiology of outdoor environment 41 4.2. Microbiology of indoor environment 43 4.3. Correlation between microbes and various meteorological factors 45 4.4. Prevalence of Aspergillus in various microenvironments 47 4.5. Air sanitation 51

5. DISCUSSION 52

5.1. Outdoor microbial concentration 52 5.2. Indoor microbial concentration 58

5.3. Meteorological studies 67 5.4. Prevalence of Aspergillus in various microenvironments 68 5.4.1. Immunological studies 71 5.5. Air sanitation 75

6. SUMMARY 77

REFERENCES

1. INTRODUCTION

The study of airborne microorganisms has expanded from the traditional arena of transmission of disease via the respiratory route to include not only human pathogens but also plant pathogens, opportunistic and non-pathogenic organisms, and aerosolized microbial by- products. Air-borne culturable and non-culturable bacteria, saprophytic fungi, free-living parasites, viruses and algae that may result in adverse health effects or environmental impact are now studied within the field of aerobiology. Particles of biological origin are generally referred to as “bioaerosols”. These are ubiquitous in nature and may be modified by human activities. Bioaerosols range in size from submicroscopic particles (<0.01ȝm) to particles >10 ȝm in diameter.

Air serves as a mode of transport for the dispersal of bioaerosols from one location to another. The composition and concentration of the microorganisms comprising the bioaerosols vary with source and the dispersal in the air until deposition (Salem and Gardener, 1994). Biologically derived materials are natural components of indoor and outdoor environments. But under certain circumstances, biological agents may be considered contaminants of outdoor. Among the various sources of outdoor air pollution, microorganisms are considered to be the most complex and the least investigated. A wide variety of microorganisms constitutes the airspora of any region and these are dispersed from the source by various agents. The transport and ultimate settling of bioaerosols are affected by its physical properties and the environmental parameters that it encounters while it is airborne. The size, density and shape of the droplets or particles comprise the most important physical characteristics, while the magnitude of air currents, relative and are the significant environmental parameters (Pedley, 1991; Lighthart, 2000). The incidence patterns of airborne particulate matters differ from place to place and season to season.

Environmental factors influence the survival of airborne microorganisms and affect their ability to colonize on surfaces after deposition while harsh environmental conditions tend to decrease the numbers of viable airborne organisms; there is variability in survival between groups of microorganisms and within genera. In general, fungal spores, enteric viruses and amoebic cysts are somewhat resistant to the environmental stresses encountered during transport

1 through the air. Bacteria and algae are more susceptible, although bacterial endospores (e.g., Bacillus spp.) are quite resistant.

Among different classes of microorganisms, fungi and bacteria have been studied most frequently as risk factors of diseases and other health effects in the work and living environment. Fungi produce large amount of spores, which easily become airborne, thus constituting an important component in microbial . The external environment is the chief source of fungi found in indoor air and seasonal variations in climatic conditions are therefore responsible not only for variations in the number and types of microbes in outdoor air but also the air indoors. The estimation of airborne microorganisms in indoor air is important for use as an index of cleanliness for any particular environment and to determine the relation they bear on human health (Jaffal et al., 1997). Most naturally occurring bacteria do not cause human illness or other complaints. The risk of illness from environmental bacteria increases only when they enter buildings in inappropriate number or multiply indoors.

In recent years, indoor air quality has become an important issue, because of the occurrence of the “” and the fact that most people spend more than 80% of their time indoors (NRC, 1981). Indoor fungal contamination depends on numerous factors including moisture, ventilation, temperature, and organic matter present in building materials but so on outdoor fungal load (Medrela – Kudar, 2003). Although indoor environments are considered to be protective, they can become contaminated with particles that present different and sometimes more serious risks than those related to outdoor exposures, when their concentrations exceed recommended maximum limits. These are 1000 cfu/m3 for total number of bio- particles set by the National Institute of Occupational Safety and Health (NIOSH), and by the American Conference of Governmental Industrial Hygienists (ACGIH) with the culturable count for total bacteria not to exceed 500 cfu/ m3 (Cox and Wathes, 1995; Jensen and Schafer, 1998).

Infectious diseases arise from microbes and involve the transmission of an infectious agent from a reservoir to susceptible host through airborne transmission. Various bacterial diseases such as, legionellosis and tuberculosis are linked to cause significant public health concern due to their low infectious dose (Stetzenbach, 2002). Legionella become airborne often as a result of active aerosolizing processes (aeration of contaminated water) and may inhabit

2 various aquatic environments including man-made water systems, often in biofilms in cooling towers, air condition system, etc. The transmission of tubercle bacilli occurs through the inhalation of aerosolized bacilli in droplet nuclei of expectorated sputum from positive tuberculosis patients during coughing, sneezing and talking. The transmission of anthrax bacilli occurs due to inhalation of the spores of Bacillus anthracis and outbreaks are often linked to bioterrorism that are spread through intentionally contaminated mail, apart from occupational exposures (Traeger et al., 2002). The airborne transmission of bacterial endotoxins of gram negative bacterial cell wall are potent pyrogens, capable of causing fever in very low concentrations (Parillo, 1993). However, the significance of commonly isolated airborne bacteria in offices, schools, residences, and outdoor environments has not been determined. This is due in part to the isolation of numerous Gram-positive cocci and Gram-negative bacilli in the absence of adverse health effects. Species of Staphylococcus and Micrococcus are commonly disseminated from nasal and oral surfaces, skin clothing and hair of building occupants. High ratios of the number of air-borne bacteria isolated from indoor air to the number isolated from outdoor air have been used as an indication of high occupancy rate, poor ventilation or inadequate building maintenance.

Since, the population spends about 80% of time indoors, there is considerable concern about the possible effect on health caused by excessive exposure to mould. These effects can be classified as infections, irritations, allergies and toxic effects (Bornehag et al., 2001, 2004).

The health and wellbeing of the public are affected by the physical, chemical and biological properties of the indoor environment. The quality of the indoor environment, however is not easily defined or readily controlled, and can potentially place human occupants at risk. Indoor air contains large numbers of airborne microorganisms. Their estimation is important for use as an index of cleanliness for any particular environment (Williams et al., 1956), and to determine the relation they bear on human health (Jaffal et al., 1997). Knowledge of the incidence of airborne micro flora in houses is important for their possible correlation to infectious diseases or associated allergic reactions (Fink et al., 1971). The concentrations of both spores and their volatile metabolites may become significantly higher in indoor, than in outdoor environments. Since people spend most of their time indoors, they are in continuous contact with the air spores and toxins, and persistent exposure may become significant even if the toxin

3 concentrations are low. Indoor microbiological pollutions have only recently received attentions, previously afforded to outdoor even indoor chemical pollutions (Yunginger et al., 1976). This is partly due to the broad array of microbial diversity that can evoke human responses, and due to the wide variations in residential, commercial and public buildings (Gammage and Kaye, 1985). Another factor contributing to the lack of concern is the difficulties encountered in sampling biological aerosols and the evaluation of data obtained and identifying related health effects encountered.

Air borne bacteria and fungi can be the cause of a variety of infectious diseases as well as allergic and toxic effects. These microbes have been studied most frequently as risk factors of diseases and other health effects in the work and living environment. Especially fungi produce large amount of spores, which easily become air borne and are able to colonize indoor environments which can utilize nutritional sources and moisture available in indoor materials (Burge, 1992; Flanningan, 1992). Though, indoor spaces with low humidity and characteristic air movements as a result of heating and natural ventilation do not provide favorable conditions for the survival of fungi (Reiss, 1991). In case of sufficient humidity, however, fungi may grow on almost all organic substances. Conditions of above 70% relative humidity may be optimal for fungal growth (Burge, 1985). Although indoor environments are considered to be protective, they can become contaminated with particles that present different and some times more serious risks than those related to outdoor exposures, when their concentration exceeds recommended maximum limits. More than 80 genera of fungi have been associated with symptoms of respiratory tract allergies (Horner et al., 1995). Alternaria, Aspergillus, Cladosporium and Fusarium are amongst the most common allergenic genera. Metabolites of fungi are also believed to irritate the respiratory systems.

The fungal spores in both indoor and outdoor environments can be studied quantitatively or qualitatively using samplers. The spectrum of indoor airborne mold spores, such as in homes, offices and other work places, differ from place to place due to the influx of spores from outdoor air through ventilations and air exchangers. Hence, it is difficult to arrive at any significant conclusion on the role of the spore in the allergic response. Again, it is not always the quantity but, allergenicity of the mold, which determines the overall development of clinical allergy. Building and behavior related problems in indoor environments may lead to massive

4 growth of mold with in a very short period of time. In recent years, the increasing incidence of allergy is well recognized not only in the developed but also in the developing countries across the globe. The likelihood that a given individual will develop an allergic disease reflects a combination of genetic and environmental factors.

Association between Immunoglobulin E (IgE) antibodies against molds and occurrence of immediate type I hypersensitivity reaction have been found among residents, and measurement of IgE antibodies as a markers of exposure is recommended in investigations of Type I hypersensitivity reactions (Chowdary et al., 2003 and Portney et al., 2005). Long term surveillance of ambient exposure levels and the monitoring of health impact in occupants by periodic examinations are the best approaches for preventing the occurrence of respiratory diseases and allergies. Preventive modification in the general environment is much more difficult but recent successful efforts to limit cigarette smoking as a passive indoor exposure agent, indicate that the future good health of our citizens mandate such government intervention.

In summary, interest in the populations of airborne microorganisms in agricultural and industrial settings, healthcare facilities, residences, offices and classroom environments has increased in recent years. The potential for adverse environmental and human health effects resulting from indoor and outdoor bioaerosols exposure has prompted renewed interest in aerobiology, and research activity in this area of environmental microbiology has rapidly expanded. Prevention of allergic contact dermatitis is possible only by avoidance or strict barrier protective measures. These recommendations are the only effective means of preventing respiratory diseases because pulmonary disease impairment may be severe and permanently disabling, early intervention and removal from the places mandatory. Symptomatic treatment for respiratory symptoms should only be recommended as a stopgap measure until the exposure is terminated.

In view of the above considerations, the present study was carried out to evaluate the quality of air, breathe whether outdoor or indoor with the following objectives;

x To collect air samples from various microenvironments of extramural and intramural air to analyse and quantify the ambient air quality outdoor and indoor in the selected locations of Chennai city, Tamil Nadu, South India.

5 x To conduct a regular investigation on the bacterial and fungal counts, percentage distributions of organisms, types of isolates, the current prevalence, seasonal and geographical variations etc., in Chennai, South India and to correlate the outdoor and indoor air quality in terms of bacterial and fungal populations. x To explore the influence of eco – physical factors such as temperature, relative humidity, rainfall and direction of air current in the microbial distribution of air. x To screen for the presence of common microbial types, their total counts, percentage distribution and seasonal variations so as to evaluate the relation between the microbial air quality and allergic status of the selected individuals in the sampling sites. x To study the total immunoglobulin E antibody level as the serological index to relate with the allergic status. x To evaluate the relationship between the specific immunoglobulin E antibodies against the indoor related microbes as biomarkers of exposure in clinical investigations of allergic reaction. x To evolve a suitable method to remediate the high risk sites by appropriate air sanitation method and check its efficacy.

6

2. REVIEW OF LITERATURE

Systematic studies on the microbiology of the atmosphere were started at the end of nineteenth century in the expectation of finding the source of epidemic diseases such as cholera and typhoid. Miquel (1850-1922) made intensive studies on the bacteria and moulds in the atmosphere at the observatory Montessori’s in Paris. He developed techniques that enabled him throughout the last quarter of the nineteenth century, to analyze daily the microbial content of outdoor air as part of long term survey of the microbial content of the atmosphere. Some of the outstanding contributions of this period were Pasteur (1861) and Miquel (1883) in France, Thomson (1854), Airy (1874) in England and Cunningham (1873) in India. However, it was during this period that BlackLey (1873) a Manchester physician, proved by inhalation experiments on him and others that hay fever is caused by inhalation of pollen. He also demonstrated that pollen at times was present in the air in large quantities. Inhaled fungal spores were recognized as allergens following the work of Cadham (1924) and Feinbery (1935) in North America. Further, the recognition of the spread of the rusts diseases of cereals by airborne uredospores greatly stimulated plant pathologists to undertake intensive aerobiological investigations (Stakman and Christensen, 1946). 2.1. Bioaerosols A collection of airborne particles is called bioaerosols. Generally, bioaerosols are generated as poly dispersed droplets or particles, consisting of different sizes ranging from 0.5 to 30ȝm in diameter (Lighthart, 1994). Air serves as a mode of transport for the dispersal of bioaerosols from one location to another. The composition and concentration of the microorganisms comprising the bioaerosols vary with the source and the dispersal in the air until deposition (Lynch and Poole, 1979). The transport and ultimate settling of bioaerosols are affected by its physical properties and the environmental parameters that it encounters while it is airborne. While the size, density and shape of the droplets or particles comprise the most important physical characteristics, the magnitude of air currents, relative humidity and temperature are the significant environmental parameters (Lighthart and Mohr., 1987; Pedgley, 1991; Lighthart, 2000) that influence the survival of airborne microorganisms and affect their ability to colonize on surfaces after deposition. In general, fungal spores, enteric viruses and amoebic cysts are somewhat resistant to the environmental stresses encountered during transport through the air. Bacteria and algae are more susceptible, although bacterial endospores (e.g.,

7

Bacillus spp.) are quite resistant (Knudsen and Spurr, 1987). Although it is generally believed that microbial numbers do not increase during transport, a doubling of airborne bacterial cell numbers were demonstrated in a rotating – drum aerosol chamber with saturated humidity conditions and tryptone added to the cell suspension prior to aerosolization (Grinshpun et al., 1997). Inhalation, ingestion and dermal contact are routes of human exposure to airborne microorganisms, but inhalation is the predominant route that results in adverse health effects. The average human inhales approximately 10 m3 of air per day (Lynch and Poole, 1979). Large airborne particles are lodged in the upper respiratory tract (nose and nasopharynx) (Zeterberg, 1973). Particles < 5 ȝm in diameter, are removed by sneezing and blowing or wiping of the nose, and those particles deposited in the pharynx (2 to 5 ȝm in diameter) are removed from the pharynx by mucociliary action and then swallowed (Brown et al., 1950; Stole, 1976). Particles 1 to 5 ȝm in diameter can be transported to the lung, but the greatest retention in the alveoli are the 1 to 2 ȝm particles (Satter and Ijaz, 1987; Salem and Gardner, 1994).). Exhaled droplets of healthy people have been measured to be from 0.3 to 8 μm although few drops were > 2 μm (Papineni and Rosenthal, 1997). Asthma, hypersensitivity pneumonitis and other respiratory illness are associated with exposure to bioaerosols (Strachan et al., 1990; Sorenson, 1999). Bioaerosols also may result in the spread of plant diseases, loss of agricultural productivity and deterioration of building materials. 2.1.1. Sources of bioaerosols in outdoor environments Numerous anthropogenic activities serve as the origin of bioaerosols in outdoor environments, especially agricultural practices and waste water treatment processes. Increases in airborne concentrations of microorganisms during harvesting have been documented by Lighthart, 1984 and Abdul Hafez et al., 1990. Presence of airborne bacteria and viruses resulting from waste water treatment were also reported (Ran ball and Led better, 1966; Lundholm, 1982). Sanitary lands fill operations (Rahkonen et al., 1987), reuse of waste water for irrigation practices (Teltsch and Katzenelson, 1978; Applebaum et al., 1984), and recycling facilities were also generate bioaerosols (Reinthaler et al., 1999). The release of biotechnology products (e.g., genetically engineered microorganisms and microbial pest control agents), agricultural productivity, mineral recovery, oil spill cleanup and toxic waste disposal can also be a source of airborne microorganisms. Aerosolized, genetically modified cells have been monitored in a barn setting (Marshall et al., 1988) and at biotechnology

8 based fermentor (Juozaitis et al., 1994). Infectious and toxogenic biological agents released for warfare or terrorism under favorable meteorological conditions may also result in severe illness for military and civilian populations (Wiener, 2000). 2.1.2. Sources of bioaerosols in indoor environments Deterioration of building materials, offensive odors and adverse human health effects are associated with microbial contamination of indoor environments. Residences, offices, school, healthcare facilities, enclosed agricultural structures, pharmaceutical and industrial facilities, food processing plants and recycling facilities are among the indoor environments where airborne microorganisms have been studied. Sources and reservoirs of microorganisms were present within these settings, including building materials and furnishings, pets, plants, and systems (Lighthart and Stetzenbach, 1994; Gravesen et al., 1999). Bacteria and algae generally grow in the areas of withstanding water such as air handling – system components and sites where water intrusion or leaking (e.g., flooding and condensation) has occurred. However with the exception of viruses, which require a living host cell for replication, microorganisms will colonize virtually any surface where there is sufficient moisture. Fungi, which have lower water activity (aw) requirements than other microorganisms, tend to colonize a wide variety of building material (Grant et al., 1989). Aspergillus versicolor and Penicillium spp. are the primary colonizers on wallpaper and drier margins of wetted walls, while Cladosporium spp. proliferates as secondary colonizers. Components of heating, ventilation and air conditioning (HAVAC) systems may serve as amplification sites and these systems have been associated with the dispersal of contaminants indoors (Buttner and Stetzenbach, 1993; Fisher et al., 1999). Naturally ventilated buildings are also affected by bioaerosols, as organisms can be transported via drafts through open windows and doors (Lighthart and Stetzenbach, 1994). When microbial amplification occurs, the indoor environment then becomes a source of bioaerosol exposure for the occupants. This exposure may be important because people spend approximately 22 h/day in indoor environments (Spangler and Sexton, 1983). Coughing and loud talking are reported to release approximately 105 droplets per m3 with a mean droplet size of >1 ȝm (Papineni and Rosenthal, 1997). Microorganisms are also dispersed from surfaces as a result of activity by the occupants (Reynolds et al., 1990; Buttner and Stetzenbach, 1993). Indoor environmental quality complaints with the description of random, vague symptoms by building occupants, such as nasal, eye and mucous membrane irritation, lethargy, headache, rashes, dry

9 skin and shortness of breath, have been termed sick building syndrome (Kiers and Hodgson, 1984; Finnegan and Pickering, 1986). A variety of possible causes of sick building syndrome include comfort factors (Temperature and humidity) (Jaakkola and Heinonen, 1989), environmental tobacco smoke (Gold, 1992), chemical off – gassing from building materials (e.g., adhesives, paints and particle board) (Weschler et al., 1992) and microbial contamination (Dales et al., 1991; Harison et al., 1992; ACGIH, 1999). Although no regulations regarding microbial contamination or bioaerosol concentrations are mandated for residential, office, or class room environments, a variety of quantitative standards and guide lines predicted on base line data have been proposed by Rao et al., 1996. It is generally accepted that indoor sources of bioaerosols may be significant when differences are noted between indoor and outdoor concentrations and/or populations (Reynolds et al., 1990) but the lack of exposure/dose response data has precluded the establishment of bioaerosol threshold limit values (ACGIH, 1999). Bioaerosols are either injected into the atmosphere by chance (e. g., wind, rain and bursting bubbles) or processes governed by natural selection. Wickmann (1994) presented an explanation based on the physical and biological (molecular process) parameters and used the term deposition, adhesion and the release to explain the transport of bioaerosols. Cox (1987) referred to the same mechanisms as “take – off processes” and ‘landing on surfaces”, that geared more to the consequences as they relate to the human respiratory system. Chamberlain (1967) studied both the deposition and release of spores and pollens as they relate to biological surfaces. 2.2. Air borne microorganisms The environmental effects and human health complaints resulting from airborne microorganisms have renewed interest in a wide variety of microorganisms. The discovery of Legionella pneumophila as the cause of the outbreak of Legionnaires disease in Philadelphia in 1976 increased the level of awareness of diseases caused by bacterial aerosols (Winn, 1988; Gold, 1992). The increased level of reporting of tuberculosis in both developing and industrialized countries has prompted renewed interest in the genus Mycobacterium and its airborne transmission. Mycobacterium tuberculosis is spread via aerosols from an infected person and is recognized as a significant public health concern because of the low infectious dose (Kaufmann and Van Embden, 1993). Nontuberculosis Mycobacteria have also been associated with respiratory illness (Kirschner et al., 1992). Aerosol dispersal of bacterial pathogens as weapons of mass destruction (e. g., Bacillus anthracis) can also result in illness and

10 death in exposed populations (Wiener, 2000). However, the significance of commonly isolated airborne bacteria in offices, schools, residences and outdoor environments has not been determined. This is due in part to the isolation of numerous Gram – positive cocci and Gram – positive bacilli in the absence of adverse health effects. Micrococcus spp. and Staphylococcus spp. are commonly disseminated from nasal and surfaces, skin, clothing and hair of building occupants (Favero et al., 1966). High ratio of the number of airborne bacteria isolated from indoor air to the number isolated from outdoor air have been used as an indication of high occupancy rate, poor ventilation, or inadequate building maintenance (Gallup et al., 1993). Exposure to airborne bacterial cells may also result in the inhalation of endotoxin, a lipopolysaccharide found in the cell wall of Gram – negative bacteria and blue – green algae. Exposure to endotoxin and respiratory impairment and endotoxin can exacerbate asthma (Rylander and Fogelmark, 1994). Airborne endotoxin may be a major cause of illness in enclosed agricultural settings such as silage facilities, poultry processing houses and cotton mills (Rylander and Vesterlund, 1982) and waste handlers have reported increased nausea and gastrointestinal problems that were associated with endotoxin exposure (Ivens et al., 1999). The International Commission on Occupational Health, through its Committee on organic dusts, suggests that endotoxin exposures may provoke different reactions such as organic dust toxic syndrome, bronchi constrictors, mucous membrane irritation, depending on the concentration and duration of exposure to the endotoxins (Reylander, 1997). A few studies of endotoxins effects on lung function have been conducted. In a study of 410 grain workers and 201 postal workers, grain workers were found to have a significantly higher prevalence of work-related and chronic respiratory symptoms than postal workers (Schwartz et al., 1995). Higher concentrations of endotoxins in the air however were associated with diminished measures of airflow and enhanced bronchial activity. Bulk and airborne samples of the environment can be tested for the presence of endotoxins and quantification of the exposures can be used to associate the levels of endotoxins with acute and chronic effects on pulmonary function. The commonly accepted method of analyzing endotoxins in environmental sample is the chromogenic modification of the Limulus amoebocyte lysate (LAL) test. The latest generation of the technique, the kinetic chromogenic modification, combines the accuracy, reproducibility and sensitivity of the chromogenic technique with the methodology to overcome sample – induced enhancement or inhibition of the test.

11

Viruses can become airborne through the release of contaminated liquids or dried material. Wind-blown carriage of animal pathogenic viruses has been shown to cause outbreaks of disease, considerable distances downwind from the source (Christensen et al., 1993; Maragon et al., 1994). For example an epizootic pseudorabies in swine herds arose from airborne spread of virus across an area of nearly 150 Km2 (Grant et al., 1994), and retrospective studies of similar outbreaks indicated airborne spread of the virus up to 17 km (Banks, 1993). Intercontinental transport of human viruses through atmospheric dispersion of airborne particles has also been postulated by Hammond et al., (1989), who suggested that such long distance transport of airborne viruses may explain the pandemics of influence. Inhalation of air with viral particles can lead to their retention in the respiratory tract, and airborne spread has been clearly documented for a variety of viral infections of humans (Sattar and Ijaz, 1987; Eickhoff, 1994) and animals. Infection through the inhalation and retention of droplet nuclei are generally regarded as true airborne spread. Infectious viruses or their nucleic acids (Sawyer et al., 1988) may be due to the translocations and ingestion of particles retained in the upper respiratory tract (Slote, 1976). In general the airborne spread of viruses is rapid as well as difficult to prevent (Casal et al., 1999). Activities such as sneezing, coughing, flushing toilets and changing chippers, as well as shaking, homogenization and sonication of virus – containing materials can generate infectious aerosols (Sattar and Ijaz, 1987). Preventing the generation of, and avoiding exposure to such aerosols is particularly important in laboratories and other settings where infectious material is handled (Cole and Cook, 1998). According to Pike (1979), 27% of the cases of laboratory – acquired infections were due to airborne viruses; cases in research settings accounted for more than 67% of such infections. While improvements in the design and constrictions of biohazard containment equipment (Clark, 1995) and better enforcement of bio safety procedures (Richmond and Mc Kinney, 1999) have considerably reduced the risk of aerosol exposure, many laboratory workers do not appear to be fully aware of the dangers of infectious aerosols. Investigations of chicken pox outbreaks clearly showed airborne spread of Varicella – zoster virus (VZV). Sawyer et al. (1994) found VZV DNA in air samples from rooms housing patients with zoster or chicken pox, and the air remained positive up to 24h after patient discharge. Whereas none of the VZA DNA – positive samples had infectious virus, PCR technology prove to be very useful in studying the airborne transport of viruses in indoor and outdoor settings. Hepburn and Brooks (1991) have described an outbreak of chickenpox in a

12 military field hospital. Numerous human viruses are transmitted via droplets and spread by the respiratory route from one person to another in the indoor environment (Zeterberg, 1973). Enteric virus bioaerosols are produced at sewage treatment facilities (Adams et al., 1982), and aerosol transport of pathogenic viruses from infected plants has also been documented (Graham et al., 1977). Serious illness and death following exposure to Hanta virus aerosolized from rodent feces and urine have been reported by Diglisic et al. 1999. Airborne viruses continue to be a threat to human and animal health in spite of the sophisticated design and efficient protective functioning of the respiratory system (Skerrett, 1994). Now, greater emphasis is needed on reducing the generation of infectious aerosols in indoor as well as outdoor settings and in enhancing the removal and inactivation of viruses (Clark and Scarpino, 1996) and other infectious agents in the air. Airborne fungi have been the focus of much concern because of their ability to cause serious respiratory infections and to elicit allergic reactions. A large number of fungi survive as saprophytes. Many of these saprophytes have been found to grow in the indoor environments of buildings and have caused building related complaints and illnesses (Miller, 1992; Samson 1992; Miller, 1993; Gravesen et al., 1994). It must be emphasized that fungal spores and occasionally hyphal fragments are dispersed and disseminated in air from one location to another. The majority of this literature is based on air – sampling data, including literature focusing on hospitals and health care facilities (Steifel and Rhame, 1993), on residential dwellings (Strachan et al., 1990; Burge, 1995; Dekoster and Thorne, 1995), on schools (Dungy et al., 1986; Levetin et al., 1995) and on office buildings (Morey et al., 1984). The focus of hospital sampling has often been on Aspergillus fumigatus, an opportunistic pathogen. General fungal populations have been identified in nonhospital samplings. A comprehensive assessment of fungal contamination in the indoor environment should include consideration of environmental factors (such as outdoor air, ventilation mode, heating, occupant density, ventilation rate and moisture), on-site inspection, air sampling, surface and source sampling, sample analysis, risk analysis and finally remedial actions (Burge, 1990). Both sexual and asexual spores of five major classes of fungi (Myxomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes) have been isolated and reported from air. Spores of Ascomycetes and Basidiomycetes have frequently been recovered from air samples by using spore trap samplers. Levetin (1991) reported 18 genera of basidiospores from the atmosphere in Tulsa, Okla. Furthermore, many species of basidiospores

13 have been demonstrated to be allergenic (Santilli et al., 1985; Benaim – Pinto, 1992). The majority of airborne fungi collected on samples and grown on agar media are Deuteromycotina and Zygomycetes. Many of these spores are known as allergens. Some of them have been prepared into allergen extracts and approved by the U.S. Food and Drug Administration for medical uses (Smith, 1990). Common Deuteromycotina found in air include Alternaria, Cladosporium and Epicoccum. Mucor and Rhizopus, both of which are Zygomycetes are also frequently isolated from air. Outdoor airborne fungal populations may directly or indirectly affect the indoor populations, since the pathways of are often suspected to be leaks and creaks or through doors, windows and building air intake systems. Therefore, common fungal taxa are often the predominant fungal types detected in indoors (Lewis et al., 1994; Yang, 1995).Yang et al. (1993) examined cultures of over 2,000 Andersen samples collected outdoors and in non residential buildings in the United States, found that Alternaria, Aspergillus, Cladosporium and Penicillium were the top fungal taxa most frequently found in indoors as well as outdoors. All the fungal taxa were detected in less than 40% of indoor samples. However, Cladosporium was found in over 80% of outdoor samples, while Penicillium was detected in 58%. These data suggest that both Cladosporium and Penicillium are common in outdoor air. It is believed that fungi actively growing indoors are the primary cause of adverse health effects due to exposure to indoor fungal allergens, mycotoxins and fungal volatile organic compounds. It is, therefore important to identify growth and amplification sites of fungi in indoors. Raper and Fennell (1977) reported various species of Aspergillus from building materials such as wall paper and paper products, textiles, jute, insulation materials and fabrics. Many species of the genus Penicillium, commonly detected in indoor air sampling are frequently referred to as agents of food spoilage and biodeterioration (Samson et al., 1994). Gravesen et al. (1994) have recorded a list of 13 fungal species as important molds in damp buildings. Samson et al. (1994) have described 23 common fungal species in indoor environments. Morgan – Jones and Jacobsen (1988) studied moldy carpets, plasterboard, and wallpaper from three hotels in Florida and Georgia. Their brief literature review suggests that many fungi had been reported to cause biodeterioration of paper, textiles and plaster. Fungi have been known to grow in heating, ventilating and air-conditioning (HVAC) systems. Yang (1996) examined and cultured 1,200 fiberglass insulation liners from HVAC systems in the United States and found fungal colonization and growth in approximately 50% of the samples studied.

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Fungi are mostly known to cause infections of the skin and other body organs, as well as allergies. Fungi and their by-products, such as (1-3) –ȕ -D-glucan, mycotoxins and VOCs have also been implicated in adverse health reactions and other diseases (Burge and Amman, 1999; Rylander, 1999). Few fungi actually cause infections in healthy individuals. Immunosuppressed host defenses resulting from organ transplantation, cancer therapy (e.g. antibiotics, steroids and drugs), or the presence of another disease – causing agent may increase the likelihood of fungal infection (Mishra et al., 1992). More commonly, exposure to fungi can initiate adverse health effects in the absence of infection by serving as aeroallergens, resulting in exacerbation of asthma, allergic rhinitis, and respiratory distress (Lacey and Crook, 1988). Exposure to Rhodotorula rubra was reported as the cause of extrinsic allergic alveolitis (Siersted and Gravesen, 1993), while Aspergillus and Penicillium have been identified as risk factors for asthma and atopy respectively in children (Garrett et al., 1998). Children exposed to fungi in damp houses are also at higher risk for both upper and lower airway symptoms and systemic symptoms of headache, fatigue, joint pain and fever have been reported by occupants of moldy environments (Rylander and Etzel, 1999). Immuno compromised patients may be at an increased risk for opportunistic infections if pathogenic fungi become airborne and are significantly elevated in indoor air surveys. Among the fungi of concern are Aspergillus spp., such as A. fumigates, A. flavus and A. niger are commonly occurred. Many fungi are known to cause an immune pathology with an exaggerated or inappropriate immune response, called hypersensitivity reactions or common allergy. The fungal spore is a known cause of allergic diseases (Horwitz and Bush, 1997; Chapman, 1999) and has been identified as one of the major indoor allergens (Pope et al., 1993; Burr, 1999). Normal or typical indoor molds may vary depending on climate variations and geographical regions. However, as mold levels that are atypical in the indoor environment increase because of recurrent water leaks, home dampness and high humidity, the prevalence of allergy and respiratory problems also rises (Burge, 1990; Dales et al., 1991; Flannigan et al., 1991). The reported percentages of population allergic to molds vary from 2 to 18%. Approximately 80% of asthmatics have been reported to be allergic to molds (Flannigan et al., 1991). The incidence and prevalence of allergic diseases are on the rise (Pope et al., 1993). Many patients with chronic rhinosinusitis have a very high incidence of positive fungal cultures (up to 96%), and chronic rhinosinusitis are often associated with allergic fungal sinusitis (Ponikau et al., 1999).

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All fungi may be allergenic depending on the exposure situation and doses (Pope et al., 1993), although the sensitivity of clinical tests may vary with the study population and individual immune system characteristics. Atopic individuals typically have a higher rate of positive skin reactions after provocation tests and serological allergy tests measuring antibody precipitins (e.g., IgE). Diseases such as allergic bronchopulmonary aspergillosis (Kurup, 1999) and allergic fungal sinusitis possibly require additional host factors which are not well documented (Raper and Fennell, 1977), and they may be the combined result of an allergenic inflammation reaction and a response to the immuno toxic effect of fungal metabolites. The relevant route of exposure is inhalation. In general, the adverse effects of fungal inhalation are related to duration and intensity of fungal exposure. However, typical for allergic reactions is that once an individual develops an allergy to certain fungi, even small air borne concentrations can trigger an asthma attack or other allergic reactions. Allergy “threshold levels” to common mold have been reported (Gravesen, 1979), but variations in sampling strategies and methodological limitations make these levels very unreliable in practical settings (Dillon et al., 1999), Therefore, the consensus is that acceptable safe threshold limits for fungal indoor exposure cannot be established (Ammann, 1999; Matcher et al., 1999), and it is generally recommended to avoid or minimize unnecessary fungal indoor exposures (Anonymous, 1994). In clinical allergy, patients can be tested for specific mold allergy using skin or serological tests e.g., immunoglobulin E (IgE), radio allergosorbent test (RAST), and appropriate advice and treatment can then be prescribed. Due to the low sensitivity of some of the commercially available mold extracts tests, false-negative results are not uncommon. Patients with an atopy are frequently allergic to multiple fungal species and manifest type –I reactions (Asthma, Rhinitis, Eczema and Hay Fever). Some fungi have been known to produce secondary metabolites called mycotoxins that, when ingested (Marasas and Nelson, 1987), inhaled (Miller, 1992; Johanning et al., 1993), or in contact with the skin (Schiffer, 1990; Dill et al., 1997), are harmful to animals and humans. These mycotoxins belong chemically to alkaloids, cyclopeptides and coumarins (Griffin, 1993). There are more than 200 mycotoxins produced by a variety of common fungi according to the World Health Organization, (1990). Advanced fungal exposure characterization and sampling techniques now available should improve the chances for better medical detection of mycotoxicosis.

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Fungi in active growth produce VOCs, which typically are noticed by their musty, moldy odor. Indoor measured VOC levels however are typically low, and possible health risks are uncertain. Measurement of VOCs may be an indicator of excessive indoor fungal growth (Keller et al., 1999). A number of VOCs have been identified from fungi common in indoor contamination. Most of these fungal VOCs are derivatives of alcohols, ketones, hydrocarbons and aromatics, the 2-ethyl hexanol and cyclohexane are eye and skin irritants and benzene is generally recognized hazardous chemical. Other fungal VOCs associated with two common indoor fungi, Aspergillus spp., and Penicillium have been identified. Almost all the published information regarding fungal VOCs concerns Aspergillus spp., Penicillium spp., and little is known about VOCs of other common indoor fungal contaminants. Risk assessment of human exposure to these fungi and their by-products is complex, because multiple agents, hypersensitivity reactions and disease outcomes are involved. Human sensitivity to these allergens varies from individual to individual. It is prudent to avoid any unnecessary exposure to infectious, allergenic and toxic fungi and to control indoor growth conditions. Spores of Streptomycetes from damp, moldy houses have been shown to increase the levels of production of inflammatory mediators (Hirvonen, 1997). Indoor exposures to thermophilic actinomycetes and resulting disease have been reported, with a and home (Hugenholtz and Fuerst, 1992) implicated as the sources. Micropolyspora faeni and Thermoactinomyces vulgaris are thermophilic actinomycetes that have been reported in association with hypersensitivity pneumonitis and other allergic reactions (Land et al., 1991). Blowing dust was reported as the source of 62 genera of airborne algae (Brown et al., 1964) and eutrophic lakes are contributors to outdoor airborne algal concentrations. Dust and aeration of fountains and aquariums (Lighthart and Stetzenbach, 1994) have been proposed as possible indoor sources while exposure to algal extracts has been associated with adverse human health effects; the extent of allergic reactions due to algal bioaerosols has not been fully investigated. Additional research is needed to determine the environmental and human health effects of airborne algae. Free-living amoebae (e.g Acanthamaobae, Naegleria fowleri) are indigenous to soil and water and can be aerosolized from natural and artificially heated waters from lakes and hot springs. Airborne Acanthamoebae species have been detected in the nasal passages of children in

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Africa (Lawande et al., 1979) and have been observed in air samples from Mexico City (Rivera et al., 1987). Humidifiers and ceiling dust were as indoor sources of airborne amoebic antigen (Edward, 1980). Although several health effects can be cited by exposure to these organisms, insufficient information on airborne protozoa is available. Universal to the field of aerobiology is the need for measurement methods for the detection and identification of the microorganisms of interest (Griffiths and DeCoseno, 1994). In general, interest in the population of airborne microorganisms in agricultural, industrial settings, healthcare facilities, residences, offices and classroom environments has increased in recent years. The potential for adverse environmental and human health effects resulting from indoor and outdoor bioaerosols exposure has promoted renewed interest in aerobiology and research activity in this area of environmental microbiology has rapidly expanded. 2.3. Sampling methods The objective of bioaerosol sampling is the efficient removal and collection of biological particles from the air in a manner, which does not affect the ability to detect the organisms, (e.g., alteration in culturability or biological integrity). This ability is dependent on the physical and biological characteristics of the organisms and on the physical features of the sampling instrument (Willeke and Baron, 1993). Impaction separates particles from the air stream by utilizing the inertia of the particles to force their deposition on to solid or semisolid surface. This process depends upon the inertial properties of the particle, such as size, density and velocity and on the physical parameters of the impacter such as the inlet nozzle dimensions and airflow pathway (Nevalainen et al., 1993). Liquid impingement is similar to impaction in that the inertial force of the particle is the principle force removing it from the air (Willeke et al., 1998). However the collection medium is a liquid, usually a dilute buffer solution and the collected microorganisms move around freely in the bubbling liquid. As a result, aggregates of cells may be broken apart and particles remaining in the air stream may diffuse to the surface of a bubble and be transferred to the collection buffer in this manner. Filtration achieves the separation of particles from the air stream by passage of the air through the porous medium, usually a membrane filter. Collection of particles depends on their physical properties (size, shape and density), the filter pore size and the air flow rate (Lee and

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Ramamurthi, 1993). Inertial forces and other mechanisms such as interception, diffusion and electrostatic attraction result in the collection of particles on the surface of the filter. Gravity is a non-quantitative collection method in which an agar medium is exposed to the environment and airborne organisms are collected primarily by gravity. Though it is inexpensive and easily performed method, collection of airborne by this method is affected by the size and shape of the particles and by the motion of the surrounding air (Nevalainen et al., 1993). As a result, large particles are more likely to be deposited on to the collection surface (Burge and Solomen, 1987). In addition, the airborne concentration of the microorganisms cannot be determined by gravity sampling because the volume of air from which the particles originate is unknown. 2.3.1. Sampler types Pouchet (1860) was the first to construct an aeroscope consisting of a funnel shaped tube carrying a glass slide coated with an adhesive, and this was later modified by Maddox (1870) and Cunningham (1873). While BlackLey (1873) introduced gravity slide sampler, early methods were summarized by Cunningham (1873). Developments during the next seventy years were reviewed by the committee on Apparatus in Aerobiology of the National Research Council, Washington. Among these, the reviews on aerobiological methods of sampling fungal spores by Lacey and Venette (1995) on outdoors air sampling techniques, and by Mullins and Emberlin (1997) on sampling in indoor are noteworthy. Some of the more widely used bioaerosol sampling methods are discussed. 2.3.1.1. Impactor sampler Impaction is the most commonly used method of collection for airborne microorganisms, and a variety of impactor samplers are commercially available. They differ by the number and the shape of the nozzles and the number of stages. If air is drawn through a single nozzle, the nozzle is usually rectangular and the impactor is referred to as a slit sampler. If there are several nozzles that resemble a sieve then, the impactor is sometime referred to as a sieve sampler. If there are several stages with successively smaller nozzles, the sampler is referred to as a cascade impactor. The Anderson six stage impactor samplers consist of six stages with decreasing nozzle diameters so that successive stages collect progressively smaller particles (Andersen, 1958). One and two stage models of the sampler are also available. The Surface Air System (SAS) and Burkard portable air samplers are battery powered one stage impactor which utilize agar filled

19 plates as the collection medium. The Reuter centrifugal sampler (RCS) and RCS plus (Biotest Dianostics corp., Denville, N.J.) are portable battery powered samplers which centrifugally impact the microorganisms onto agar strips. The portable samplers do not require external vacuum pumps and electrical outlets but are available only as single stage devices. Slit impactors deposit the bioaerosol onto an agar surface are used for the estimation of viable cells. (e.g. Casella MK - JL distributed by BGI, Inc., Waltham, Mass) or onto an adhesive coated surface for the microscopic enumeration of the collected particles, usually fungal spores or pollen grains (e.g., Burkard spore trap, and Air – O - Cell sampling cassette, Zefor International, Inc., St Petersberg, Fla.). Many slit impactors have a moving collection surface to provide temporal discrimination of the bioaerosol concentration. 2.3.1.2. Liquid impinger sampler These are commonly used for the retrieval of bioaerosol particles over a wide range of airborne particle concentrations. Sample for analysis, the liquid sample can be concentrated by filtration or diluted by liquid addition, depending on the concentration of microorganisms. The liquid samples may also be analyzed by biochemical, immunological and molecular biological assays to detect the presence of specific microorganisms, culturable or non culturable. The AGI – 30 all glass impinger sampler (Ace Glass, Inc., Vineland, N.J.,) is a widely used liquid impinger sampler that has a curved inlet tube designed to stimulate the nasal passage, making this sampler useful for studying the respiratory infection potential of bioaerosols (Grinshpum et al., 1994; Jensen et al., 1994). The inlet tube is washed with a known volume of collection fluid to recover non respirable airborne particles. The AGI – 30 has an impaction distance of 30 mm from the jet to bottom of the sampler. The AGI – 4 model features a shorter distance of 4 mm to improve particle collection efficiency over that of the AIG – 30. The Burkard multistage liquid impinger is a stainless steel sampler which collects particles in three size fractions: >10, 4 to 10, and < 4 ȝm. The recently developed biosampler (SKC, Inc. Eighty four, Pa.), which combines impingement into a liquid with centrifugal motion (Willeke et al., 1998) can be used for viscous collection fluids (e.g., heavy white mineral oil). Having the same inlet geometry and the same airflow rate (12.5 L/minute) as AGI samplers, the biosampler achieves particle collection by drawing aerosol through three nozzles that are directed at an angle toward the inner sampler wall. During normal operation the liquid swirls upward on the sampler’s inner wall and collected particles are removed.

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2.3.1.3. Filtration sampling The method of collection of airborne microorganisms onto a filter material is used in bioaerosol monitoring due its simplicity, low cost and versatility. Air samples are usually collected onto 25, 37, or 47 mm diameter filter membrane housed in disposable plastic cassettes, which are available from a variety of manufacturers (e.g., Gelman Sciences, Ann Arbor, Corning Coster Corp., Bedford, Mass; Nucleopore, Corning Colster Corp., Cambridge, Mass and SKC Inc). Polycarbonate, cellulose mixed ester or polyvinyl chloride filter material may be used depending on the nature of the bioaerosol and the method of sample analysis (Lee and Ramamurthi, 1993; Jenson et al., 1994). Filter membrane pore size range from 0.01 to 10 μm with air sample flow rates from 1 to 50 Liters/minute. This sampling is adaptable to a variety of arrays, but loss of viability of vegetative cells may occur, presumably due to desiccation stress during sampling (Jensen et al., 1992; Thorne et al., 1992). The MD 8 air sampler (Sartorius AG, Goettingen, Germany), collects airborne microorganisms on a gelatin filter to reduce desiccation stress. The gelatin membrane is incubated on the agar medium of choice for culture analysis. 2.3.2. Sampler performance Data from these studies are often difficult to compare because of differences in samplers, the length of sampling time, the volume of air sampled, the sample analysis method and the characteristics of the bioaerosol being measured. The performance of bioaerosol samplers can be divided into physical and biological components. Depending on the external wind direction and velocity relative to the inlet geometry and flow characteristics, particle concentration measurements, which are higher or more commonly lower than the true concentration in the environment, may be obtained (Brockmann, 1993). Collection efficiency is the ability of the sampler to remove particles from the air stream and transfer them to the collection medium. Biological sample efficiency differs among bioaerosol sampler types. Effects of particle bounce and reaerosolization as well as the evaporation of the collection fluid are minimized in the biosampler, which can operate with non - evaporating and highly viscous liquids (Willeke et al., 1998). The overall design of the biosampler’s collection unit minimizes the impaction stress. When used with heavy white mineral oil, the biosampler can maintain microbial viability and high physical collection efficiency for several hours (Lin et al., 1999; Lin et al., 2000).

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2.3.3. Collection time It is an integral part of the bioaerosol sampling design and guidelines for the selection of optimal sampling times for various bioaerosol samplers have been published (Navalainen et al., 1993; Macher, 1999). For each sample, the sampling period must be sufficiently long to obtain a representative sample of the airborne microorganisms present without exceeding the upper quantization limit of the sampler or causing losses in culturability of air borne organisms. Short air sample periods provide only a brief temporal and spatial glimpse of the environment and several samples may be required to determine the average bioaerosol concentrations. One way to predict the optimal sampling time for a particular sampler is to determine the ideal surface density of microorganisms on the collection area and assume the order of magnitude of the bioaerosol. Nevalainen et al. (1992) calculated the optimal sampling time for five bioaerosol samplers by using the formula t= (į) (A)/ (a) (Q), where t is the sampling time, (į) is the desired surface density, (A) is the area of the sampling surface, (a) is the average expected bioaerosol concentration and (Q) is the sampler flow rate. A doubling time of sampling time may not result in a doubling of cfu, depending on the stress tolerance of the airborne microorganisms being sampled. Therefore, the investigator should consider making several consecutive short sample collections rather than taking a few samplers over a long interval. 2.4. Culture methods Many of the currently available bioaerosol sampling methods rely on culture for the quantification and characterization of airborne bacteria and fungi. It is necessary to perform replicate sampling with different media or to divide samples for inoculation onto multiple types of nutrient media. Several conditions, such as temperature, pH, water activity, nutrients, antibiotics, light and aeration can be manipulated to favor the growth of select group of organisms. The growth of organisms on selective media is hindered compared with that on general media. This may be especially true when airborne microorganisms, which are already stressed or damaged by aerosolization and sampling, are being cultured. For this reason, general media are often used for the initial culture of bioaerosol samples, and then replication onto differential or selective media for identification is performed (Jensen et al., 1994). For the culture of bacteria several broad spectrum media, such as tryptic soy agar, nutrient agar, and casein soy peptone agar may be used. (Jensen et al., 1994; Burge, 1995). These media are often amended with antibiotics to restrict the growth of fungi. Incubation from 28Û C to 35Û C for 1 to

22

7 days are usually used for environmental and human source bacteria, an important exception being thermophilic actinomycetes, which are cultured at 55Û C. Several broad spectrum media has been evaluated for culturable airborne fungi (Burge et al., 1977; Verhoeff et al., 1990). Malt extract agar, rose bengal - containing agar and dichloran glycerol – 18 agar have been suggested for isolation of airborne fungi. Other commonly used fungal media include Potato dextrose agar and Sabouraud’s dextrose agar (Soloman, 1975). Incubation periods for fungi typically range from 3 to 7 days; most airborne fungi are mesophilic and grow well at temperatures of 20 to 25 ÛC and medically important fungal pathogens grow well at 30Û C (Madelin, 1995). Incubation temperature can also be used to select certain species. The concentration of culturable airborne microorganisms (cfu/m3) is determined by dividing the number of cfu per sample by the volume of air sampled. When microbes are collected with multiple jet impactor samplers (e.g., Andersen and SAS impactor samplers), positive hole correction are generally applied to the data (Andersen, 1958; Buttner and Stetzenbach, 1993). As colony counts increase, counting errors increase because of overlap of colonies and inhibitory effects of microorganisms on one another. For statistical accuracy, microbiologists traditionally use data only from plates with colony counts between 30 and 300 per 100 mm diameter plate. More than one sample collection is needed for better enumeration. Microscopic methods are used to obtain an estimate of the total number of microorganisms present in a sample. A variety of stains maybe utilized to differentiate biological particles from non - biological material and respiring cells from non respiring cells. Accurate identification to the genus level is possible for a limited number of fungal spore types. Therefore, data are usually reported as total number of spores per cubic meter. For the determination of airborne bacteria by microscopic analysis, liquid impingement or filtration sampling is used. Cells and endospores may also be enumerated by bright - field or phase – contrast microscopy using a hemocytometer or counting chamber. Misidentification of fungal spores and the inability to distinguish microorganisms from non - biological particles are common sources of error with this method (Burge, 1995). 2.4.1. Immunoassays Immunoassays rely on the binding of antibodies to a specific target antigen. Target antigens may be (i) cell surface associated proteins or polysaccharides or (ii) human allergens. Among the methods which may be applied to bioaerosol analysis are fluorescence immunoassay, enzyme immunoassay and radioimmunoassay. Enzyme immunoassay utilizes binding of the

23 antibody or the antigen to an enzyme. The concentration of antigen is measured by enzyme activity and in radioimmunoassay by radioactivity. These methods have been applied for the measurement of airborne allergens, such as dust mite allergen, animal dander and Beta - 1, 3 – glucan (De Blay et al., 1991; Platts – Mills et al., 1992). The advantages of these methods for quantification of airborne allergens are their specificity and sensitivity. The major limitation, of immunoassays is that specific antigens for microorganisms are difficult to define and standardize (Madelin and Madelin, 1995). As more microbial antigenic compounds are characterized, immunoassay analysis methods will provide data necessary for assessment of environmental exposure to allergens. Biochemical assays are also used to measure biological compounds of interest, such as endotoxins or mycotoxins. The most widely used method for measurement of endotoxin is the Limulus amebocyte lysate (LAL) test. This reaction forms the basis of endotoxin quantization and a variety of test systems are commercially available. Filtration sampling with glass fiber filters or polycarbonate membranes has been used to measure aerosolized mycotoxins of Stachybotrys chartarum in the laboratory (Pasanen et al., 1993) and in the field (Yike et al., 1999). Other biochemical methods which may be applied to the analysis of airborne fungal exposure include ergosterol (Miller and Young, 1997) and ȕ – 1, 3 - glucan (Fogelmark et al., 1992; Douwes et al., 1996) assays. 2.5. Indoor air quality surveys: The professional approach No other area in occupational and environmental health has experienced such rapid changes in the recent past as has indoor air quality. Due to an emphasis on energy conservation, buildings are more dependent on mechanical ventilation systems for the delivery of fresh air. Analytical recommends a common sense approach to indoor air quality investigations. Analytical has consistently endeavored to maintain its approach to the assessment and control of indoor air pollution problems on the leading edge of technology. A routine program of building inspection and assessment can identify problems before compliance and building – related symptoms occur. This is reflected in the growing body of regulatory requirements and guidelines, as described below. (i) Occupational Safety and Health Administration Standards (OSHA) proposed Indoor Air Quality Rule, 29 CFR 1910, 1915, 1926 and 1928. (ii) Environmental Protection Agency (EPA): Guide for building owners and facility managers.

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(iii) American Conference of Governmental Industrial Hygienists (ACGIH): Guidelines for the assessment of bioaerosols in the indoor environment. (iv)National Institute of Occupational Safety and Health (NIOSH): Guidance for indoor air quality.

The air handling systems are visually inspected for microbiological contamination on various system components. Upon completion of our visual investigation, the associated ventilation assessment, measurement of comfort parameters, microbiological testing for volatile chemical compounds, a report is generated. The recommended range for humidity is 30% to 60%. ASHRAE (American society of Heating, and Air conditioning Engineers Standard) recommends that temperature be maintained at 69º to 75 ºF in the winter and 73 º to 79 º F in the summer, with a relative humidity in the range of 30 – 60 %. National Institute of Occupational and Health (OSHA) guidelines, call for sufficient fresh air to maintain carbon dioxide concentrations below 2,500 ppm. More recent indoor air quality data indicate that in order to prevent human discomfort, carbon dioxide concentrations should not exceed 1,000 ppm. Ventilation measurements are therefore, often conducted to determine the amount of fresh air delivered to each occupant of the building. ASHRAE has recommended supplying fresh air at a minimum rate of 20 cubic feet per minute (cfm) per building occupant for the normal office building. Volatile Organic Compounds (VOC) vaporizes at and pressure. They can cause eye, nose and throat irritation, headache, nausea and possible damage to the central nervous system. Total VOC air samples are collected in accordance with NIOSH method 1500/1501, using charcoal sorbent tubes that are analyzed by gas chromatography with flame ionization detection. Persistent or intermittent water and humidity breeds microbiological contamination. HVAC systems with moist, warm conditions enhanced by the presence of dust and dirt make great homes for microorganisms. High humidity provides excellent breeding areas for these microbes. The airborne concentrations of bacteria and fungi are reported separately in units of colony forming units per cubic meter of air (cfu/m3).

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2.6. Efficacy data and labeling requirements of Air sanitizers Requirements apply to products with label claims for the treatment of air to reduce the numbers of air borne microorganisms. Glycol vapors produce significant decreases in numbers of viable air borne bacteria when properly and continuously dispensed by a vaporizing device so as to maintain suitable concentration in the air of enclosed spaces. Several investigators have shown that glycols (triethylene, dipropylene, or propylene glycol) at concentrations of 5% or more in such formulations will temporarily reduce numbers of airborne bacteria when adequate amounts are dispensed under relatively ideal conditions (Robertson et al., 1943 and Mc Gray, 1970).

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3. MATERIALS AND METHODS

3.1. Sampling site

Chennai is the fourth most populous metropolitan area and the fifth most popular city situated at Latitude 13° 04’ N and Longitude 86° 17’ E on the Southeast coast of India. Chennai metropolitan area extends over 1180 sq km and has a population of more than 8.5 million. Rapid urbanisation with vehicle congestion has increased menacingly on the roads of Chennai. As a result of this, gaseous pollutants and respirable suspended particulate matter pollutants are continuously increasing in the ambient air, consequently an increase in the incidence of respiratory allergic diseases are being reported in Chennai.

3.2. Survey protocol

The present study measured the bacterial and fungal concentrations in the outdoor and indoor air of various microenvironments under uncontrolled environmental conditions from January 2007 to December 2008. Samples from each microenvironment were surveyed for bacteria and fungi throughout the study period. An effort was made to geographically disperse the sampling sites throughout the city. It was expected that the summer would provide a more favourable humid environment for microbial growth, resulting in different bioaerosols levels. The indoor and outdoor air measurements were taken concurrently or consequently at each sampling site. The majority of the indoor air measurements were taken from the middle of the facility or living area at breathing height, while the outdoor air measurements were taken from outside area of the surveyed facility.

The microenvironments selected from outdoor were Bus stand, Railway station, Recreation ground, Sewage treatment plant and Vegetable market and indoor such as Home, Hotel, Office premise, Public toilet and Theatre.

3.3. Schedule of air sampling

The air sampling was performed during regular morning (between 10am – 12 noon) and regular evening hours (between 17:00h and 19:00h) on week days (Monday to Friday). Homes selected from typical residential areas were surveyed and each residence was occupied by a single family with three to six persons. All the households participated

27 throughout the study periods. At the same time, no homes reported the use of a dehumidification system during sampling period. Most of the office premises and hotels were located on the ground level and were surveyed throughout the revise episode. There were staff members (between 5to20) working in the office that accepts many visitors right through the day. For comparison purposes, measurements were also made, the periods of direct sunlight were minimal because of the closed environment was recorded. This increases the probability of survival of air borne microorganisms as the sun’s direct UV radiation can reduce the viability of many microorganisms. However, the microenvironment of theatre samples was collected from the rear of the theatre to minimize the interruption, and the sampling was performed during the show time and intermission times to examine bioaerosols levels. The same survey was also performed in public toilets.

A total of 10 microenvironments (5 from each environment) were selected for the study. From each microenvironment, 12 samples were surveyed at regular intervals of a month for the study period. Each sample was a mean of triplicate, which was collected once in 10 days intervals.

3.4. Air sampling methods

3.4.1. Sampling device

The sampling device, the Air Petri Sampling System Mark II (Hi media laboratories limited, India) is highly specialized instrument devised to collect and to enumerate of sieve impactor as described by Anderson (1958) which aspirates air through a perforated plate. The instrument consists of a powder coated aluminium container with clamp designed to accommodate a petridish containing a nutrient agar or any other desired medium. The unit consists of powder coated tall stand, adaptor for mounting (either vertical or horizontal direction), SS cone (sampling system) for 90 mm petriplate and SS feeder cone circular clamp with SS feeder cover. The impactor stage contains 340 precision drilled holes. When air is drawn, the air borne particles are impinged towards the surface of the agar. The diameter of the orifice is 1.5millimeter. The schematic picture of a sampler as a whole is shown in figure 1 and the sampler is operated at the flow rate of 100 L/min. The portable sampler is operated by a remote system using chargeable batteries.

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Figure 1. Hi – Air Sampling System

3.4.2. Preparation of the sampler

Sampler fractionating units were sterilized in hot air oven at 160°C for 1 h before using. Prior to the sampling process the fractionating units were swabbed with cotton and dipped in 70% ethyl alcohol and samplings were taken only after the evaporation of alcohol. The autoclaved SS cone was fixed in the sanitized sampler; petriplate of 90 mm diameter was kept in the sampler with 20 ml of culture medium which serves as collection surface. The plate resets on the raised metal pins. Lid of the petriplate was removed and immediately the plate was covered with autoclaved SS feeder cone circular clamp assembly in position. The device was visually checked to ensure a good sealing.

3.4.3. Air sampling

The sampler was placed centrally within each microenvironment and was raised to a sampling height of approximately 1 meter. The agar plate with its lid removed was placed on the base of the sampler, so that the plate rests on raised metal pins. Immediately the plate was covered with the SS feeder cone with SS feeder cover and the device was secured tight and

29 visually checked for good seal. The cover of the unit is perforated, with a perforation of predetermined size. A vacuum pump draws a known volume of air through the SS feeder cone with where it is accelerated and passes onto the stage, and the particles in the air containing microorganisms impinge on the agar medium in the petridish. The exhaust air is then carried through the outlet on the base and relaxed outside.

After sampling, the inlet cone was released and the agar plate was removed. The agar plate cover was replaced quickly and labelled at the back with the appropriate sampling and identification information. The sampler was resanitized with 70% ethanol wipe between each sample location and allowed to dry. All exposed plates were transported to the laboratory for incubation.

Nutrient agar, Blood agar, MacConkey agar and Potato dextrose agar (Appendix I) were used for the enumeration, isolation, and propagation of different microbial communities. The exposed petriplates containing media were sealed with parafilm and transported to the laboratory for incubation. The plates were incubated at 37° C for 24 – 48 h for bacterial growth and at room temperature (28 ± 2° C) for 48 – 72 h for the isolation of mesophilic fungi. The counts for the air sample plates were corrected for multiple impactions using the positive hole conversion method (Anderson, 1958) and reported as colony forming units per cubic meter (cfu/m3) of air. The colonies were then identified and subcultured for further studies.

3.5. Identification of bacteria

The isolated bacteria were identified through microscopic examination, biochemical testing using Cowan and Steel’s manual for the identification of bacteria (Cowan, 1974).1974;

3.5.1. Preliminary tests

3.5.1.1. Gram staining (Christian Gram, 1884)

The organism on culture was stained using Gram staining method. The smear preparation, staining and fixation, decolourization and counter staining were carried out conventionally and observed under the microscope for bacteria.

3.5.1.2. Motility test

Clean cavity slides along with cover slips were taken for each isolate. A drop of culture suspension was placed at the centre of the cover slip and vaseline was applied to the

30 corners of the cover slip, which was then placed over the cavity slide. The slide was then viewed microscopically to check the motile nature of isolates under 40X.

3.5.1.3. Catalase test – slide method

Pure culture of the isolate was transferred from the agar to a clean slide with a loop or glass rod. Immediately, a drop of 3% hydrogen peroxide was added to the culture and observed for effervescence. Negative and positive controls were kept for confirmation.

3. 5. 1.4. Oxidase test

Oxidase enzyme plays a vital role in the operation of electron transport system. This test depends on the presence of certain oxidases in bacteria that catalyse the transport of electrons between electron donors in bacteria and a redox dye tetramethyl – p – phenylenediamine dihydrochloride. The dye is reduced to a deep purple colour. Oxidase disc was placed on a clean glass slide which was then placed in the petridish. The dish was moistened with distilled water. The colony to be tested was picked up using a tooth pick and smeared over moist area. Then the colour development was observed.

3.5.2. Biochemical tests

3.5.2.1. Indole production test

The ability to hydrolyze tryptophan with the production of indole is dectectable by adding Kovac’s reagent composed of p – dimethyl aminobenzaldehyde which produces a cherry red colour. Sterile peptone broth, 5 ml was inoculated with the culture and incubated at 37° C for 48 h. Following incubation, 0.2 ml of kovac’s reagent was added to observe the colour change.

3.5.2.2. Methyl red test

Sterile glucose broth, 5 ml was inoculated with the test culture and incubated at 37° C for 48 h. Following incubation, 5 to 6 drops of methyl red solution was added to detect the ability of microorganisms to oxidize glucose with the production and stabilization of high concentrations of acid end products.

3.5.2.3. Voges – proskauer test

This test is to determine the capacity of some organisms to ferment carbohydrates with the production of non-acidic or neutral end products. Sterile glucose broth, 5 ml was inoculated with the test culture and incubated at 37° C for 48 h. Following incubation 1ml of

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40% potassium hydroxide and 3 ml of 5% solution of alpha naphthol in absolute ethanol were added to observe the colour change.

3.5.2.4. Citrate utilization test

Some microorganisms use citrate as the sole carbon source and grow. Citrate is acted upon by the enzyme citrase which produces oxaloacetic acid and acetate. These are then enzymatically converted to form products. This reaction is shown by the change in the colour of the indicator. The medium was streaked with broth culture of test organism and incubated at 37° C for 24 h. After incubation period, colour change was noted.

3.5.2.5. Triple Sugar Iron (TSI) agar test

TSI test is used to differentiate different groups of Enterobacteriaceae according to their ability to ferment lactose, sucrose and glucose and the production of hydrogen sulphide. The fermentation reaction of the sugars will help to distinguish organisms. The TSI slants contained 1% each of lactose, sucrose and glucose in a concentration of 0.1%. The phenol red, the acid base indicator was incorporated in the medium to detect carbohydrate fermentation. The medium was inoculated with the test culture by first stabbing the butt down to the bottom and then streaking the surface of the slant and incubated at 37° C for 24 h to observe the colour change.

3.5.2.6. Urease test

Urease is an enzyme produced by few microorganisms. Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds like urea and forms alkaline end products such as ammonia. Production of urease is detectable when the organisms are grown in urea broth medium containing phenol red, pH indicator which shows the change in colour. This medium was inoculated with the test culture and incubated for 24 h at 37° C and the colour change was noted.

3.5.2.7. Carbohydrate fermentation test

Most microorganisms obtain their energy through a series of orderly and integrated enzymatic reaction leading to the bio - oxidation of a substrate, frequently a carbohydrate. In fermentation, substrates such as carbohydrates and alcohols undergo anaerobic dissimilation and produce an organic acid that may be accompanied by gases such as hydrogen or carbon

32 dioxide. The broth was inoculated with the test culture and incubated at 37° C for 24 h and observed for gas production.

3.5.2.8. Oxidative fermentation test

This method depends upon the use of a semisolid medium containing the carbohydrate (usually glucose) together with a pH indicator. If acid is produced only at the surface of the medium, where conditions are aerobic, and the attack on the sugar is oxidative. If acid is found through out the tube, including the lower layers where conditions are anaerobic, the breakdown is fermentative. Duplicate tubes with medium were inoculated by stabbing. One tube was covered with liquid paraffin to a depth of 5 – 10 mm and both were incubated at 37° C for 48 h or longer and observed for acid production.

3.6. Identification of fungi

The exposed petriplates containing Potato Dextrose Agar (PDA) sealed with parafilm were transported to the laboratory for incubation. The plates were incubated at room temperature (28 ± 2° C) in the glass chamber for the isolation of mesophilic fungi. After incubating for a period of 2 to 3 days, the colonies were counted, identified and subcultured for further studies.

3.6.1. Morphological characteristics

3.6.1.1. Macroscopic appearance

Morphological characteristics of the culture viz, colour, shape, pigmentation, reverse pigmentation were studied by using the hand lens.

3.6.1.2. Microscopic appearance

Microscopic characteristics were studied by preparing the slides and observing under light microscope. The characters of conidia bearing structure, shape, size, separation, colour and ornamentation were observed. Lactophenol with cotton blue (for hyaline molds) was used for staining fungi. The prepared slides were sealed with DPX (Qualigens fine Chemicals) for preservation. The fungi were identified with the help of standard manuals and monographs (Gilman, 1957; Moubasher, 1993) which is based mainly on gross colony appearance and microscopic examination of the spore and mycelium.

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3.7. Presentation of data 3.7.1 Air sampling (Kalogerakis et al., 2005) The colonies of individual organisms were converted to number/m3 of air by multiplying with a factor calculated as follows and the counts are expressed as colony forming units (cfu)/cubic meter of air (m3).

Suction rate of the sampler = 100 L/min

Duration of each sampling = 4 min

Amount of air sampling in 4 minute = 4*100 = 400 L/m3

Let the number of colonies recorded = X

Thus the number of colonies recorded /m3 of air = 1000 /400* X

The conversion factor = 2.5

3.7.2. Isolation frequency

The term isolation frequency has been used to denote the number of sampling in which an organism was recorded as against the total number of samplings (in each microenvironment). On the basis of percent isolation frequency the organisms were grouped as, Most common = above 80% and 100%

Common = 60% and below 80%

Frequent = 40% and below 60%

Occasional = 20% and below 40%

Sporadic 1% to below 20%

3.7.3. Percent contribution

The term percent contribution refers to the contribution of individual organism to the total and is calculated as follows

Percent contribution = No. of cfu/m3 of an individual organism/ Total no. of cfu/m3 of all organisms

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3.8. Recording of meteorological parameters

Monthly mean temperature (°C), mean relative humidity (%), mean total rainfall (mm) and mean wind speed (km/h) were obtained from the Regional Meteorological centre, Government of India, Chennai. Geographic distribution, seasonal variation and annual trend of organisms were studied in detail.

3.9. Statistical analyses

The statistical analyses were performed using mini tab version and excel 2007 on a personal computer. Analysis of variance (ANOVA) was used for the comparison of data sets with monthly mean temperature (° C), mean humidity (%), mean rainfall (mm) and mean wind speed (km/h). Both Pearson’s correlation co – efficient and Spearman’s rank correlation co – efficient were calculated between different meteorological factors with aerosol concentration in each microenvironment during the sampling periods. A paired t – test was employed for the comparison of the bioaerosol data sets of the outdoor and indoor air. Meanwhile, a non parametric test (Mann – Whitney test) was employed for the comparison of the bioaerosol data sets of five different microenvironments each from outdoor (bus stand, railway station, recreation ground, sewage treatment plant and vegetable market) and indoor air (home, hotel, office premise, public toilet and theatre) and two sampling periods. The mean and standard deviation (SD) were used to characterise the normally distributed data. The criterion for significance in the procedure was P<0.05.

3.10. Immunological analysis

The present work investigated the indoor air quality in terms of fungal population and to evaluate between the microbial air quality and the allergic status of the selected individuals in the sampling site to study the immunoglobulin E antibody as the serological index to relate with the allergic status.

3.10. 1. Study design

Allergy is more common in urban areas when compared to rural areas. This clearly suggests that air population may play a possible role in allergic diseases. The relation between the microbial air quality and the allergic status of the selected individuals was evaluated by Enzyme Linked Immuno Sorbent Assay method. The most common invitro test for assay of allergen activity is ELISA (Wisdom, 1976; Turner et al., 1980).

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This study was an incident case – control study. A total number of 115 blood samples were collected from the volunteers of the selected occupants with an age group of 21 – 64 years for the case study and a total of 25 samples were collected from the healthy individuals for control study living in the microenvironment (homes), a geographically defined industrialized and highly polluted area (Chennai). Recruitment of controls took place at regular intervals throughout the study period.

3.10.2. Selection of cases

Volunteers were selected from the microenvironment (homes), first in the city from January 2009 to August 2009, based on a questionnaire, to be used in general population, (Jaakkola and Miettinen, 1995; Jaakkola and Jaakkola, 1999) included: (i) health information (ii) active smoking (iii) occupation and work environment (iv) home environment and (v) dietary questions. As an additional route of case selection, a history of at least any one asthma symptoms such as, prolonged cough, wheezing or nocturnal cough was included for the study. All the confirmed cases of asthma like symptoms fulfilling the general eligibility criteria were selected for the case study. A total of 115 blood samples were collected for eosinophil counts, total IgE and specific IgE antibody analyses.

3.10.3. Selection of controls

The controls were drawn from the volunteers of the same microenvironment and the general criteria were also applied. Recruitment of controls took place at a regular interval throughout the study period. Blood samples were collected from 25 individuals (controls) and processed immediately.

3.10.4. Screening test

3.10.4.1 Differential WBC count (Parra et al., 2000)

Neutrophils, eosinophils, basophils, lymphocytes and monocytes are the five types of leucocytes normally found in blood. To determine the relative percentage of each type of leucocytes, the blood smear will be stained and examined under oil immersion. A total of 100 white blood cells will be recorded. This method of white blood cell enumeration is called a differential count. The blood smear was prepared and air dried. Then it was stained with Leishman’s stain and examined under oil immersion objective.

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3.10.5. Estimation of total IgE

3.10.5.1. Enzyme Linked Immuno Sorbent Assay (ELISA) (Chowdary et al., 2003)

The collected blood samples were processed for the IgE level. Serum was separated by centrifugation and stored at - 20° C before shifting to the laboratory for total IgE antibody analyses. The IgE quantitative ELISA provides a rapid, sensitive and reliable assay for total serum IgE. The minimal sensitivity of this assay is about 5.0 IU/ml. The individuals with atopic allergic diseases exhibit increased total immunoglobulin E (IgE) levels in blood. In general, elevated levels of IgE indicate an increased probability of an IgE – mediated hypersensitivity.

Materials and components

x Antibody coated microtitre wells, 96 wells per plate.

x Reference standards, 0, 10, 50, 100, 400 and 800 IU/ml (liquid).

x Zero buffer (13 ml)

x Enzyme conjugate reagent (18 ml)

x Substrate TMB (13 ml)

x HCl, 2N (10 ml)

Assay procedure

The entire reagents were brought to room temperature (18 - 25° C) before use. Lyophilized standards were reconstituted with 1.0 ml distilled water, and allowed to stand for at least 20 minutes. Reconstituted standards were stored at 2 - 8° C. The desired number of coated wells was secured in the holder. Standard specimens, 20 μl of each were dispensed into appropriate wells. Zero buffers (100 μl) was dispensed into each well and thoroughly mixed for 10 seconds. The plate was then incubated at room temperature (18 - 25° C) for 30 minutes. The incubation mixture was removed by flicking plate content into a waste container. The microtitre wells were washed with distilled water 5 times, with 30 seconds soaking time. The wells were then sharply tapped on an absorbent paper to remove all residual water droplets. Enzyme conjugate reagent (150 μl) was dispensed into each well and

37 gently mixed for 5 seconds. The plate was then incubated at room temperature for 30 minutes.

The wells were washed 5 times with distilled water with 30 seconds soaking time for each wash, after incubation. Tapping on an absorbent paper was done to remove the residual water droplets. TMB solution (200 μl) was dispensed into each well and gently mixed for 5 seconds. Incubation was carried out in the dark for 20 minutes. The reaction was stopped by adding 50 μl of 2N HCl to each well and gently mixed for 30 seconds (It is important to make sure that all the blue colour changes to yellow colour completely). Then optical density was read at 450nm with a microtitre reader. Finally the absorbance of the test serum was measured and compared with standards.

Values and sensitivity

Serum IgE values are expressed in International Units/ml. Laboratories working independently have confirmed that 1 IU equals approximately 2.4 ng of protein. The total IgE level in a normal, allergy free adult is < 160 IU/ml of serum. The minimum detectable concentration of IgE by this assay is estimated to be 5.0 IU/ml. To determine the IgE level of the patient, first the mean absorbance value for reference standards was established.

3.10.6. Estimation of specific IgE (Weir(Kerr etet alal.,., 19871994 and and Brummund Pound, 1998) et al., 1987)

The estimation of specific IgE was done by Enzyme linked immunosorbent assay. In ELISA, antibodies that exhibit high binding affinities to different surface chemicals motifs of the immunogen are made use of. The method is based on the interaction of an antigen with its specific antibody.

x Coating buffer: The buffer (Carbonate – bicarbonate buffer, 100 mM, pH 9.6) was prepared by dissolving 840 mg of sodium bicarbonate and 1.06 g of sodium carbonate in 100 ml of distilled water. The pH of the buffer was adjusted to 9.6 with 0.1N HCl

x Blocking buffer: Phosphate Buffer Saline (PBS), 10 mM, pH 7.2 with 0.1% fish gelatin and 0.05% (w/v) sodium azide.

x Washing Buffer: Phosphate buffered saline - Tween (PBS – T), 0.01 M, pH 7.2 containing 0.05% (v/v) Tween – 20 and 0.01% sodium azide.

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x Substrate buffer: Diethanolamine (10% v/v) buffer, pH 9.6, containing substrate – p – nitrophenyl phosphate (1.25 mg/ml) buffer and 0.05 mM magnesium chloride (v).

x Stopping reagent: NaOH, 5N was added as a stopping reagent and it was prepared by dissolving 20 g of NaOH in 100ml of distilled water.

x Allergen extracts: Allergen to be coated on the microtitre plate was prepared by mixing 38.5 μl of allergen extract with 11.5 μl of coating buffer to get an antigen concentration of 10 μg/50 μl of buffer.

Few wells of microtitre plate were coated with 50 μl of allergen extract prepared (50 μl/well) in coating buffer. Another set of few wells were coated with coating buffer containing fish gelatine, which serves as blank. Plates were dried overnight at 37° C in an ELISA incubator. After incubation, antigen coated plates were washed six times with washing buffer. Wells were blocked for non specific binding with 100 μl blocking buffer/well and incubated at 37° C for 30 minutes. Plates were washed six times with washing buffer and 50 μl of patient’s serum/well (sera showed positivity for total IgE) was added and incubated at 37° C for three hours. Plates were then washed eight times and 150 μl of substrate buffer was added to each well, kept for incubation at 37° C for 45 minutes. Finally, 100 μl of stopping reagent (5N NaOH)/ well was added. The absorbance was read at 405 nm.

3.11. Air sanitation Test standard x No standard method for evaluating air sanitizers has been adopted. Referring to the attached references for information on testing products intended for sanitizing the air of enclosed spaces. x The quantitative microbiological assays must be performed, using an air sampling device, to show the level of reduction of viable microorganisms achieved with the product, used as directed, in an enclosed experimental room or chamber (Wolf et al., 1959).

x The methodology employed, such as spraying and sampling procedures, and the environmental conditions in the room or chamber, such as temperature, relative humidity, etc., must be reported. Interpretation of the results must be included in the reports.

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Performance standard (Mc Gray, 1970)

The results must show the adequate vapour concentrations are achieved in the air of the test enclosure. The results must also show a viable count reduction at least 99.9% over the parallel untreated control, after correcting for settling rates, in the air of the test enclosure.

A mouldy home, the risk of asthma for its residents, associated with an exposure to the total mould or to some specific genera, probably increases with the inhalation of fungal particles as well as their products. Hence, proper identification and elimination of the microbial source in occupational and house hold settings, use of filters in ventilation and air cleaning by the use of and biocides. Bacillocid (Ghosh et al., 2004) is the commonly used, commercially available surface and environmental disinfect that has very good cleansing property along with bactericidal, fungicidal, viricidal and sporicidal activities. It does not require shutdown of the contaminated areas for 24 h.

Application

The application was made in closed spaces (all doors and windows were closed; air conditioners were turned off). The prepared solution (0.5% Bacillocid) was sprayed in wash floors, other tiled surfaces taking care to cover corners and other inaccessible areas by using soft sprayer in a closed room liberally allowing a contact time of 30 minutes and allowed to dry. The temperature and relative humidity were also recorded. Precautions were made not to ventilate rooms immediately after sanitation. Fans were switched off to slow down evaporation.

Sanitized High Air Sampling System Mark II was placed centrally within the sanitized room (home) and the agar plate (Potato dextrose agar) with its lid removed was fixed. The sampling was done after the device secured tight. After sampling the agar plate was removed and kept for incubation at ambient temperature (28± 2° C) for 48 to 72 h. The sampling was done before and after sanitization to check its efficacy.

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4. RESULTS

This chapter includes the investigation related to the bacterial and fungal counts on various microenvironments of outdoor and indoor air, their percentage distributions, the type and frequency of isolates found during the study period, seasonal and geographical variations, findings related to common microbial types, evaluation of microbial air quality and allergic status of the selected individuals in sampling sites, study on total and specific immunoglobulin E antibody level to relate with the allergic status and an appropriate air sanitation method to remediate and check its efficacy.

4.1. Microbiology of outdoor environment

4.1.1. Bacteria

4.1.1.1. Concentration of bacterial population

The occurrence levels of the total bacteria identified in the outdoor air from five different microenvironments (Bus stand, Railway station, Recreation ground, Sewage treatment plant and Vegetable market; Fig.2) and their percentage are presented in Table 1. A total of 5495 cfu/m3 bacteria was obtained from outdoor environments, of which 3032.5 (55.2%) and 2462.5 (44.8%) were obtained during 2007 and 2008 respectively. During the two year study period, air samples from recreation ground were observed with highest number of bacteria (1332.5; 24.2%) followed by sewage treatment plant (1200; 21.8%), railway station (1120; 20.4%) and vegetable market (1095 ; 19.9%) . Air samples from a microenvironment of bus stand with 747.5 cfu/m3 (13.6%) were found to be least in number. The overall percentage of the total bacterial count was decreased by 10% from 55.2% in 2007 to 44.8% in 2008. However, the total bacterial count from railway station and sewage treatment plant was higher in 2008 than the previous year (2007).

4.1.1.2. Distribution of bacteria

Table 2 summarizes the month wise distribution of bacterial concentration measured in the outdoor air. Moreover, the microenvironmental difference was also observed for the outdoor air concentrations of total bacteria. The total count was noted to vary during each month in all the sampling environments and was not uniform throughout the study period. More number of total bacterial counts (cfu/m3) were observed during the month of January 315; 10.4% (2007) and

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252.5; 10.2% (2008) from outdoor air and least number was found during the month of October, 2007 (212.5 cfu/m3) and December, 2008 (160 cfu/m3).

4.1.1.3. Type and total number of bacterial isolates

The different types of microorganisms isolated from extramural air of the various microenvironments were falling under eight different genera, of which 3 and 5 were Gram positive (3125; 56.9%) and Gram negative (1510; 27.5%) bacteria respectively. Species of Staphylococcus (1160; 21.1%) and Bacillus (1017.5; 18.5%) were found to be the most prevalent among Gram positive and Gram negative bacteria respectively. Other bacteria such as species of Micrococcus, Aeromonas, Escherichia, Pseudomonas, Klebsiella and Serratia were also identified among the air samples (Fig.4). From the extramural air, total number and percentage distribution of each genus are presented in Table 3. The unidentified bacteria which contributed 860 cfu/m3 (15.7%) were also recorded. Prevalence, month wise percentage distribution and the annual load of bacteria from outdoor environment are also presented in Table 4; Figs.5-7.

4.1.2. Fungi

4.1.2.1. Concentration of fungal population

Along with the total bacteria, the current study also determined the occurrence levels of the total fungi identified and their percentage in outdoor air from various microenvironments which are presented in Table 5. A total of 4845 cfu/m3 fungi was obtained, of which 2347.5 (48.4%) and 2497.5 (51.5%) were obtained during 2007 and 2008 respectively. During the study period, air samples from a microenvironment of bus stand were observed with highest number of fungi (1045; 21.6%) followed by sewage treatment plant (1000; 20.6%). There was a slight difference between railway station and vegetable market in total fungal count which was 985 (20.3%) and 972 (20.1%) respectively. Among the five different microenvironments, a least count (842.5; 17.4%) of total fungi was obtained from recreation ground. The overall percentage of total fungi was increased by 3% from 48.4% in 2007 to 51.5% in 2008. The total fungal count from each microenvironment was higher during 2008 than the previous year (2007) except from recreation ground. In general, the concentrations of total fungi in outdoor air were lower than the total bacteria.

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4.1.2.2. Distribution of fungi

Month wise distribution of fungal concentrations measured in the outdoor air is presented in Table 6. Total fungal count 230 cfu/m3 (9.8%) was obtained during the month of January, 2007 and 267.5 (10.7%) in February, 2008. A least number of fungal counts 150 (6.4%) and 165 (6.6%) were observed during the month of August 2007 and 2008 respectively from an outdoor air. In general, the total count was observed to vary during each month in all the sampling environments and was not uniform throughout the study period.

4.1.2.3. Type and total number of fungal isolates

The total number of individual fungal isolates and their percentage distribution from extramural environment are tabulated in Table 7. A total of 9 different genera of fungal groups were identified of which Aspergillus spp. (1600; 33.0%) was found to be the most prevalent organisms. Penicillium spp. (597.5; 12.3%) and Alternaria spp. (375; 7.7%) were also found to be the second and third respectively. Organisms such as species of Cladosporium, Curvularia, Fusarium, Mucor, Rhizopus and Trichoderma were also identified among the examined groups (Fig. 9). Few types of fungal groups were not found to be identified and grouped separately with a total of 675 cfu/m3 (13.9%). Prevalence and month wise distribution and the annual load of fungi from outdoor environment are presented in Table 8; Fig.10 -12. 4.2. Microbiology of indoor environment

4.2.1. Bacteria

4.2.1.1. Concentration of bacterial population

A total of 4902.5 bacteria (cfu/m3) was obtained in the indoor air from various microenvironments (Home, Hotel, Office premise, Public toilet and Theatre; Fig.13) are presented in Table 9. Of which, 2787.5 (56.8%) and 2115 (43.1%) were obtained during 2007 and 2008 respectively. During the two year study period, a highest number of bacteria (1200; 24.5%) was obtained from home and followed by office premise (1065; 21.7%). There was a slight difference between hotel and public toilet counts with a total of 912.5 (18.6%) and 930 (19.0%) respectively. The least count of total bacteria was obtained from air samples of theatre with795 cfu/m3 (16.2%).

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The overall percentage of total bacteria was decreased by 13.7% from 56.8% in 2007 to 43.1% in 2008. The total bacterial concentrations from exposed plates are shown in Figs.3a&b.

4.2.1.2. Distribution of bacteria

Table 10 summarizes the month wise distribution of bacterial concentrations measured in the indoor air. The microenvironmental difference was surveyed in each month and noted to vary in all the sampling environments throughout the study period. A total bacterial count of 272.5 cfu/m3 (9.8%) was measured during April, 2007 and 212.5 cfu/m3 (10.0%) in March, 2008. Higher counts were also noticed during January and August of both the years. The lowest number of bacterial counts, 197.5 (7.1%) and 147.5 (7.0%) were obtained at the month of July, 2007 and December, 2008 respectively. There was a less difference between outdoor (Table.2) and indoor (Table.10) bacterial counts in month wise distribution, ranging from 10.4 to 6.5% (outdoor) and 10.0 to 7.0% (indoor).

4.2.1.3. Type and total number of bacterial isolates

A total of 9 different bacterial genera were identified, of which 3 were Gram positive (3060; 62.4%) and 6 were Gram negative (1147.5; 24.4%) bacteria respectively. Species of Staphylococcus (1302.5; 26.6%) and Micrococcus (1180; 24.1%) were the predominant bacterial groups. Other bacteria identified were species of Bacillus, Aeromonas, Escherichia, Pseudomonas, Klebsiella, Serratia and Proteus. Organisms of unidentified groups were also obtained with a total number of 695 cfu/m3 (14.1%). The total number, percentage distribution of each type of bacteria and the annual load from indoor environment are presented in Table 11 &12; Figs.14 -16.

4.2.2. Fungi

4.2.2.1. Concentration of fungal population

The occurrence levels of the total fungi obtained from an indoor air from various microenvironments are presented in Table 13. A total of 5055 cfu/m3 fungi was recorded from the indoor microenvironment, of which 2837.5 (56.1%) and 2217.5 (43.9%) were obtained during 2007 and 2008 respectively. During the study period, a highest number of fungi (1145; 22.7%) was observed from a microenvironment of home. The second highest number was obtained from theatre (1135; 22.5%) and was found to be a least difference between the first and second

44 microenvironments. Hotel was found to be the third (1005; 19.9%) in count. There was a slight difference in count between office premise and public toilet with a total of 895 (17.7%) and 875 (17.3%) respectively. The overall percentage of total fungi was decreased by 12.2% from 56.1% in 2007 to 43.9% in 2008. The total number of fungi received from an indoor environment is given in Fig.8b.

4.2.2.2. Distribution of fungi

The month wise distributions of fungal concentrations measured in the indoor air are presented in Table 14. Total fungal counts of 290 cfu/m3 (10.2%) and 230 cfu/m3 (10.4%) were obtained during January, 2007 and February, 2008 respectively. The least number of fungal concentrations such as 170 (6.0%) and 135 (6.1%) was measured for the year 2007 and 2008 respectively during the month of September in both years. More number of fungal concentrations was also received during March (267.5; 9.4%), May (262.5; 9.2%) from 2007 and January (210; 9.5%), December (212.5; 9.6%) from 2008. Similarly, the number of total fungi was found to be less in August, 2007 and June, 2008.

4.2.2.3. Type and total number of fungal isolates

The type and total number of fungal isolates were identified and grouped into 9 different genera from the intramural environment (Table. 15). A total number of 1862.5 cfu/m3 (36.8%) of species of Aspergillus and 647.5 cfu/m3 (12.8%) of Penicillium were found to be the most prevalent groups. Organisms such as species of Alternaria, Cladosporium, Curvularia, Fusarium, Mucor, Rhizopus and Trichoderma were also identified among the fungal groups (Fig 9). Unidentified groups of fungi were recorded with a total of 547.5 cfu/m3 (10.8%). The total number, percentage distribution and annual load of different species of fungal isolates are presented in Table 16; Figs.17-19.

4.3. Correlation between microbes and various meteorological factors

4.3.1. Correlation between monthly bacterial load and various meteorological factors

Both Pearson’s co efficient correlation (parametric) and Spearman’s rank correlation coefficient (non parametric) were calculated between each bacterial group (cfu/m3) in various microenvironments of outdoor air (Bus stand, Railway station, Recreation ground, Sewage treatment plant and Vegetable market) and different meteorological factors such as monthly mean

45 maximum temperature (° C), monthly mean relative humidity (%), monthly mean rainfall (mm) and monthly mean wind speed (Km/hr) for the year 2007 and 2008 (Table 17). There were no significant correlations except Staphylococci (Pearson’s r = 0.6; P< 0.05), Micrococci (Pearson’s r = - 0.7; P< 0.05) loads and wind speed in vegetable market during 2007. Staphylococci and Micrococci showed a negative correlation (Spearman’s r = -0.75; P<0.05; Spearman’s r = -0.7; P<0.05 respectively) with mean monthly rainfall during 2007 in Bus stand and Micrococci alone showed a negative correlation (Spearman’s r = -0.74; P<0.05) with mean monthly rainfall during 2007 in vegetable market.

Parametric and non parametric correlation co - efficient were performed between each bacterial species (cfu/m3 of air) in various microenvironments of indoor air (Home, Hotel, Office premise, Public toilet, Theatre) and different meteorological factors during the study period. In home environment, Staphylococci showed a positive correlation (Pearson’s r = 0.59; P<0.05) with relative humidity and a negative correlation (Pearson’s r = - 0.6; P<0.05) with wind speed for 2008. Moreover Aeromonas and E.coli showed a positive correlation (Pearson’s r = 0.65; P<0.05 and Pearson’s r = 0.6; P<0.05 respectively) with rainfall for the same year. There were no significant correlations calculated except E.coli which showed a correlation coefficient (Spearman’s r = 0.6; P<0.05) with rainfall and Staphylococci showed a negative correlation (Spearman’s r = -0.6; P<0.05) with wind speed during 2008.

In hotel, Staphylococci showed a negative correlation (Pearson’s r = - 0.77; P<0.05) with wind speed for 2008, where as in office premise there were no significant correlation except Staphylococci and Aeromonas load (Pearson’s r = 0.6; P<0.05 and Pearson’s r = 0.6; P<0.05 respectively) with relative humidity for 2008. In case of public toilet, Staphylococci showed a negative correlation (Pearson’s r = - 0.59; P<0.05), Bacillus showed a negative correlation (Pearson’s r = - 0.69; P<0.05) and Klebsiella also showed a negative correlation (Pearson’s r = - 0.77; P<0.05) with mean monthly wind speed for 2008, where as in theatre, Bacillus showed a positive correlation (Pearson’s r = 0.6; P<0.05) with mean monthly wind speed during 2007.

4.3.2. Correlation between monthly fungal load and various meteorological factors

Outdoor and indoor total fungi loads were higher during January, 2007 and February, 2008 and a lowest count was studied during the month of August (outdoor) and September (indoor) for two year (2007 and 2008) study (Table 6&14). Relative humidity above 70% supports the growth

46 of fungi and some of the fungi especially Aspergillus is able to tolerate a high temperature because of its thermo stable property. Because of these reasons, total fungi load may not have much difference in season wise as well as location wise. The mean monthly wind speed (7 Km/h) influences the fungal distribution in various outdoor and indoor environments. It is an established fact that the temperature and relative humidity are two important factors for fungal spore generation, release and dispersal; particularly in indoor environments. Higher temperature (°C), relative humidity (%), rainfall (mm) and wind speed (Km/h) (Table 17) and associated higher concentration of culturable fungi are supporting the observations of earlier researches.

Wet and humid conditions which provide suitable environment for the growth of fungi consequently increase the total load. Correlation co-efficient between different meteorological factors and fungal concentrations in outdoor air showed, there were no significant correlations in various microenvironments of outdoor air, except Alternaria which had a positive correlation (Spearman’s r = 0.60; P<0.05) with mean monthly wind speed in bus stand during the year 2007. However, Cladosporium showed a negative correlation (Pearson’s r = -0.63; P<0.05) with mean monthly wind speed in recreation ground during 2007.

Correlation co – efficient between different meteorological factors and fungal concentrations indoor showed Penicillium had a positive correlation (Pearson’s r = 0.61; P<0.05) with wind speed during 2007 in hotel environment. Similarly, Rhizopus showed a positive correlation (Spearman’s r = 0.66; P<0.05) and Aspergillus showed a negative correlation (Spearman’s r = - 0.67; P<0.05) with mean monthly wind speed in hotel (2007) and in public toilet (2008) respectively. There were no other significant correlations except Rhizopus showed a positive correlation (Pearson’s r = 0.68; P<0.05) with mean monthly temperature in theatre environment during 2007.

4.4. Prevalence of Aspergillus in various microenvironments

In recent years, there has been an increased interest in better characterizing the properties of the fungi present indoors, in analysing the influence that have a risk factor for asthma, and in determining how variations in exposure levels to fungi at home influence the risk of developing or exacerbating asthma. This study was therefore undertaken to determine the presence and seasonal distribution of the fungus considered to be most allergenic (Aspergillus) in the air.

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4.4.1. Total number and percentage distribution of Aspergillus

Total number and percentage distribution of Aspergillus in various microenvironments are shown in the Table 18. A total of 1600 cfu/m3 and 1862.5 cfu/m3 of Aspergillus were obtained in outdoor and indoor air environments respectively. Among the outdoor and indoor microenvironments, vegetable market and home environment showed the highest percentage of Aspergillus, 41.60 (405 out of 972.5) and 48.60 (557.5 out of 1145) respectively. The lowest percentage of 31.1% (262.5 cfu/m3) was obtained in recreation ground during the study period. There was no much difference in percentage distribution of Aspergillus in other microenvironments. Prevalence and the monthly concentrations of Aspergillus during 2007 and 2008 are presented in Table 19a; Figs.20&21.

4.4.2. Annual concentration of Aspergillus during 2007 and 2008.

Table 19b shows the annual concentrations (cfu/m3; Mean ± SD) of Aspergillus in various microenvironments during 2007 and 2008. Parametric (t-test) and non-parametric tests (Mann-Whitney test) were performed between the Aspergillus concentrations of 2007 and 2008 in various outdoor and indoor microenvironments. Studies of Aspergillus load with home environment between 2007 and 2008 showed that t-test and Mann-Whitney tests were significant (P<0.05). However, in hotel environment only t-test was significant (P<0.05). Other than that, there were no significant correlations with mean concentration of Aspergillus between 2007 and 2008 in any other microenvironments.

4.4.3. Immunological studies Indoor air quality has become an important health concern and susceptible persons have a high chance of response to allergens. Considering the fact that an allergic reaction may occur with exposure to minute concentration of an allergen, mold indoor could create health risk for the individuals occupying such a building. The basic investigations required in the evaluation of an individual with suspected allergic symptoms include, peripheral eosinophil percentage, and total IgE levels. Special investigation like specific IgE estimation with ELISA (Enzyme linked immunosorbent assay) is also required (Figs. 24 a&b).

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4.4.3.1. Characteristics of the study population Total IgE antibodies were analysed for 115 cases (82%) and 25 controls (18%). The characteristics of these cases and controls are presented in Table 20. A total study population included men and women who were also currently exposed to environmental tobacco smoke and reported a history of parental allergic diseases. A total of 46 male subjects (40%) and 69 female subjects (60%) were analysed for the total IgE level. Of these 5 subjects (4.3%) were reported to be parental atopic symptoms and 11 (9.6%) and 5 (4.3%), to environmental exposure to smoke (ETS). A total of 25 healthy subjects were studied as control, of these 11 (44%) and 14 (56%) respectively of male and female. Subjects who exposed to ETS were 4 (16%) and 2 (8%) from the work place and home respectively

4.4.3.2. Age wise distribution of study population

The age wise distribution of the study population for the estimation of total IgE concentration is shown in Fig. 22. Majority (37 of 115; 32.2%) of case study subjects were from the age groups ranging from 21 to 30 and from 31 to 40 years. Considerable number of middle age group subjects (25 of 115; 21.7%) was also from 41 to 50 years and a total number of 10 (8.7%) subjects were found between 51 and 60 years. Further, the total number of cases was less (6 of 115; 5.2%) amongst the aged groups who were in-between 60 and above years. A least number of control study subject (1 of 25; 4%) was also included from 60 and above year’s group. 

4.4.3.3. Gender distribution in study population

Among the cases studied for the total IgE measurement, a highest total of male (15 of 115; 13%) subjects were found to be the age group ranging between 21 and 30 years, a least number of male subject (1 of 115; 0.9%) was studied between 60 and above years. A highest number of female subjects (24 of 115; 20.9%) were observed with the age group between 31 and 40 years. A lowest of 5 female subjects (4%) was studied under the age group between 60 and above years. A highest number of male subjects (3; 12%) were found between the age group of 21 and 30, 41 and 50 years. However in female subjects, it was 5 (20%) between 41 and 50 years (Table 21) .Only one male subject (1; 4%) and no one in female subject was observed under 60 and above years.

49

4.4.3.4. Percentage of Eosinophils in subject study

Table 22 summarizes the percentage distribution of Eosinophils in subjects studied. A total number of 106 out of 115 (92.2%) showed peripheral blood eosinophil counts within the normal range (1 – 5%). About 7 of 115 (6.1%) showed an elevated percentage of eosinophil count and 2 of 115 (1.7%) had a very high eosinophil range of 8%. Blood samples collected from the control subjects (n=25) showed the eosinophil counts within the normal range. The present eosinophil count results were used for evidence of allergy in this study.

4.4.3.5. Total serum IgE levels (Mean ± SD) in different age groups of persons showing allergic symptoms and persons with no allergic symptoms

Out of 115 subjects analyzed for total serum IgE level, about 94 individuals (39 males and 55 females) showed the serum IgE ranging below 160 IU/ml. The mean ± SD analysis were done between different age groups of both sex of individuals, showing at least any one allergic symptom. There was no significant difference between the means of the different groups (P: 0.36). A total of 25 persons showing no allergic (controls) symptoms were studied for the total serum IgE levels which showed no significant difference between the means of different groups. About 11 males and 14 females were included and shown in Table.23; Fig.23. A total of 115 suspected persons were studied for total serum IgE levels, of which 21(18.3%) (7 males and 14 females) showed positive ranging above 160 IU/ml. The rest of 94 (81.7%) (39 and 55) individuals showed the IgE level below 160 IU/ml. However, a highest number of males (3) and females (5) were studied between 21 and 30 years. The highest serum total IgE was estimated ranging between 801 and 960 IU/ml which included 1 male and 2 female cases which are shown in the Table. 24.

4.4.3.6. Serum specific IgE level

Out of 21 total IgE positive cases, 3(of 115; 2.6%) of the subjects showed positivity to serum specific IgE against Aspergillus antigen (Table 25). A highest level of specific IgE was noticed as 706.8 IU/ml which included a female of age group between 21 and 30 years. Following this, 512.1 IU/ml and 482.6 IU/ml were obtained with the age group of 21 and 30 (female) and 31 to 40 (male) years respectively (Fig.25). Interpretation of serum specific IgE antibodies as bio markers of exposure is not a problem, because such antibodies last in the blood for few days which reflects the magnitude of exposure.

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4.5. Air sanitation

In the present result, fungal count has been reduced tremendously (reduced from 100% to 2%) (Fig.26b), by the application of Bacillocid (Fig.26a), a commonly used commercially available surface and environmental that has very good cleansing property. The prepared solution (0.5% Bacillocid) was sprayed in wash floors, other tiled surfaces taking care to cover corners and other inaccessible areas by using soft sprayer in a closed room liberally allowing a contact time of 30 minutes and allowed to dry. The temperature and relative humidity were also recorded. Precautions were made not to ventilate rooms immediately after sanitation. Fans were switched off to slow down evaporation. The sampling was done before and after sanitization to check its efficacy.

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Table 1. Total number and percentage distribution of bacteria (cfu/m3) in various microenvironments of outdoor air during the study period (2007 – 2008).

Year

Microenvironment 2007 2008 Total % (n=3032.5; 55.2%) (n=2462.5; 44.8%) (n=5495) Bus stand 422.5 325 747.5 13.6

Railway station 505 615 1120 20.4

Recreation ground 892.5 440 1332.5 24.2

Sewage treatment plant 590 610 1200 21.8

Vegetable market 622.5 472.5 1095 19.9

Table 2. Distribution of bacteria (cfu/m3) in outdoor air

Total number of bacteria (n=5495) 2007 2008 Month BS RS RG SP VM Total % BS RS RG SP VM Total % January 45 45 92.5 67.5 65 315 10.4 35 55 42.5 82.5 37.5 252.5 10.2 February 35 57.5 67.5 62.5 47.5 270 8.9 27.5 75 32.5 67.5 37.5 240 9.7 March 40 45 67.5 50 75 277.5 9.1 22.5 55 37.5 40 40 195 7.9 April 37.5 45 82.5 45 42.5 252.5 8.3 22.5 45 47.5 72.5 40 227.5 9.2 May 55 32.5 82.5 37.5 47.5 255 8.4 22.5 60 52.5 47.5 35 217.5 8.8 June 35 47.5 90 75 47.5 295 9.7 35 60 45 35 20 195 7.9 July 20 50 45 52.5 67.5 235 7.7 32.5 40 42.5 50 50 215 8.7 August 27.5 42.5 67.5 40 50 227.5 7.5 17.5 45 32.5 42.5 35 172.5 7.0 September 25 47.5 77.5 40 40 230 7.6 30 25 15 65 52.5 187.5 7.6 October 32.5 37.5 70 30 42.5 212.5 7.1 27.5 65 22.5 47.5 47.5 210 8.5 November 35 32.5 82.5 47.5 47.5 245 8.1 35 45 32.5 40 37.5 190 7.7 December 35 22.5 67.5 42.5 50 217.5 7.2 17.5 45 37.5 20 40 160 6.5 Total 422.5 505 892.5 590 622.5 3032.5 100 325 615 440 610 472.5 2462.5 100

Table 3. Type and total number of bacterial isolates (cfu/m3) from outdoor air (2007 & 2008)

Outdoor air (2007 & 2008) Organisms BS RS RG SP VM Total %

Staphylococcus spp. 157.5 172.5 392.5 205 232.5 1160 21.1

Micrococcus spp. 155 185 375 55 177.5 947.5 17.2

Bacillus spp. 210 180 177.5 232.5 217.5 1017.5 18.5

Aeromonas spp. 47.5 105 150 15 227.5 545 9.9

Escherichia coli 47.5 52.5 37.5 292.5 60 490 8.9

Pseudomonas spp. 12.5 42.5 35 25 77.5 192.5 3.5

Klebsiella spp. 0 0 0 95 20 115 2.1

Serratia spp. 30 25 27.5 65 20 167.5 3.0

Others 87.5 357.5 137.5 215 62.5 860 15.7

Total 747.5 1120 1332.5 1200 1095 5495 100

Table 4. Bacterial concentrations (cfu/m3 Air; Mean r SD of 12 months) in different outdoor locations during 2007 and 2008

Bus Stand Railway Station Recreation Ground Sewage Plant Vegetable Market Species 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 Staphylococcus spp. 7.5r 4.4 5.6r 4.1 6.7r 4.4 7.7 r 2.7 23.5r 8.2 9.2r 3.9 9.2r 5.4 7.9r 4.5 8.8 r 6.5 10.6r 5.6 Micrococcus spp. 7.3r 4.2rrrrrr 5.6 r 3.6 10.0 r 5.8 r r r 5.4 r 4.2 22.5r 11.8 r r r 8.8 r 5.3 rrrrr rrrrr rrrrr 2.3 r 2.5 2.3 r 2.3 8.1r 5.9 6.7 r 3.7 Bacillus spp. 11.5 r 2.7rr rrrr 6.0 r 4.8 8.3 r 7.7 r r r 6.7 r 3.7 9.0r 3.6 r r r5.8 r 4.2 r r r 9.6r 6.3 9.8 r 5.8 10.4 r 4.6 r r r 7.7 r 2.7 r r r Aeromonas spp. 2.9 r 2.8 rrrrrr1.0 r 2.0 6.7 r 5.5 r r r 2.1 r 3.0 9.6r 9.3 r r r2.9 r 3.3 0.4 r 1.0 rrrr rrrr rrrr 0.8 r 1.6 14.8r 8.3 4.2 r 3.4 E.coli 2.9 r 2.6 1.0 r 1.3 2.3 r 2.5 rrrrrr 2.1 r 2.1 2.3 r 2.7 0.8 r 1.2 12.9 r 7.0 11.5 r 6.8 2.1 r 2.3 rr rr rr 2.9 r 3.3 0.4 r 1.0 Pseudomonas spp. 0.6 r 1.1 2.5 r 2.6 1.0 r 1.3 1.9 r 1.9 1.0 r 1.3 0.6 rrr 1.1rrrr 1.5 r 2.0 3.8 r 4.1 2.7 r 2.0 Klebsiella spp. 0.0 r 0.0 0.0 r 0.0 0.0 r 0.0 0.0 r 0.0 0.0 r 0.0 0.0 r 0.0 3.5 r 3.1 4.4 r 4.0 0.0 r 0.0 1.7 r 2.5 Serratia spp. 1.5 r 1.7 1.0 r 1.3 1.5 r 2.0 0.6 r 1.1 1.5 r 2.0 0.8 r 1.2 2.7 r 2.5 2.7 r 2.5 1.3 r 1.7 0.4 r 1.0 Others 1.3 r 2.0 6.0r 3.6 4.2 r 3.9 25.6r 6.6 4.2 r 4.2 7.3 r 2.5 9.6r 5.1 8.3 r 3.1 2.7 r 3.1 2.5 r 2.8

rrrrrr rr rr rr Statistical Analysis: ANOVA; t-test; Mann-Whitney test Bus stand Concentration of Bacillus spp. in 2007 significantly (P<0.05) higher than those of other species; Concentrations of Staphylococcus spp. and Micrococcus spp .in 2007 significantly (P<0.05) greater than those of other species other than Bacillus spp.; Concentrations of Staphylococcus spp. , Micrococcus spp., Bacillus spp. and unidentified other species in 2008 significantly (P<0.05) greater than those of other species in that year.

Railway station Concentrations of Staphylococcus spp., Micrococcus spp. Bacillus spp. and Aeromonas spp. in 2007 significantly (P<0.05) greater than those of other species in that year; Concentration of unidentified bacteria (others) in 2008 significantly (P<0.05) greater than those of other species in that year.

Recreation Concentrations of Staphylococcus spp. and Micrococcus spp. in 2007 and 2008 significantly (P<0.05) greater than those of other species in ground the respective years; Concentrations of Bacillus spp. and Aeromonas spp. in 2007 significantly (P<0.05) greater than those of other species, other than Staphylococcus spp. and Micrococcus spp. in that year.

Sewage plant Concentrations of Staphylococcus spp., Bacillus spp., E. coli and unidentified other bacteria in 2007 and 2008 significantly (P<0.05) greater Vegetable market thanConcentrations those of other ofspecies, Staphylococcus in the respective spp., Micrococcus years. spp., Bacillus spp. and Aeromonas spp. in 2007 and 2008 significantly higher than those of other species in the respective years.

Table 5. Total number and percentage distribution of fungi (cfu/m 3) in various microenvironments of outdoor air during the study period (2007 – 2008)

Year 2007 2008 Microenvironment (n=2347.5; (n=2497.5; Total %

48.4%) 51.5%) (n=4845) Bus stand 495 550 1045 21.6

Railway station 470 515 985 20.3

Recreation ground 477.5 365 842.5 17.4

Sewage treatment plant 440 560 1000 20.6

Vegetable market 465 507.5 972.5 20.1

Table 6. Distribution of fungi (cfu/m3) in outdoor air

Total number of fungi (n=4845) 2007 2008 Month BS RS RG SP VM Total % BS RS RG SP VM Total % January 52.5 40 55 37.5 45 230 9.8 52.5 52.5 42.5 62.5 35 245 9.8 February 35 42.5 32.5 30 47.5 187.5 8.0 60 75 55 30 47.5 267.5 10.7 March 37.5 52.5 30 52.5 40 212.5 9.0 45 50 35 60 52.5 242.5 9.7 April 40 50 45 42.5 37.5 215 9.2 40 47.5 27.5 40 70 225 9.0 May 30 32.5 32.5 25 55 175 7.5 45 37.5 30 32.5 30 175 7.0 June 60 32.5 52.5 47.5 35 227.5 9.7 60 17.5 35 65 55 232.5 9.3 July 47.5 37.5 45 32.5 35 197.5 8.4 47.5 27.5 17.5 50 30 172.5 6.9 August 27.5 37.5 35 22.5 27.5 150 6.4 30 45 22.5 30 37.5 165 6.6 September 35 35 22.5 35 37.5 165 7.0 30 45 30 52.5 25 182.5 7.3 October 37.5 35 52.5 42.5 32.5 200 8.5 52.5 35 17.5 40 40 185 7.4 November 40 32.5 30 25 35 162.5 6.9 40 37.5 22.5 32.5 37.5 170 6.8 December 52.5 42.5 45 47.5 37.5 225 9.6 47.5 45 30 65 47.5 235 9.4 Total 495 470 477.5 440 465 2347.5 100 550 515 365 560 507.5 2497.5 100

Table 7. Type and total number of fungal isolates (cfu/m3) from outdoor air (2007 & 2008)

Outdoor microenvironments Organisms BS RS RG SP VM Total %

Alternaria spp. 102.5 50 70 122.5 30 375 7.7

Aspergillus spp. 315 317.5 262.5 300 405 1600 33

Cladosporium spp. 42.5 65 77.5 75 50 310 6.4

Curvularia spp. 80 97.5 50 00 22.5 250 5.2

Fusarium spp. 75 32.5 50 77.5 52.5 287.5 5.9

Mucor spp. 65 62.5 37.5 95 110 370 7.6

Penicillium spp. 112.5 122.5 110 107.5 145 597.5 12.3

Rhizopus spp. 65 52.5 40 42.5 42.5 242.5 5.0

Trichoderma spp. 42.5 42.5 35 00 17.5 137.5 2.8

Others 145 142.5 110 180 97.5 675 13.9

Total 1045 985 842.5 1000 972.5 4845 100

Table 8.Fungal concentration (cfu/m3 Air; Mean r SD of 12 months) in different outdoor locations during 2007 and 2008

Bus Stand Railway Station Recreation Ground Sewage Plant Vegetable Market Fungus species 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 Alternaria spp. 5.2 r 4.1 3.3 r 3.6 2.7 r 3.4 1.5 r 2.2 3.3 r 4.0 2.5 r 3.4 3.5 r 2.3 6.7rr r 3.3 2.5 r3.0 0.0 r 0.0 Aspergillus spp. 12.9 r 6.6 13.3rrrrrrrrrr r 5. 11.7 r 5.2 14.8 r 5.7 11.5 r 8.4 10.4 r 4.1 11.7 r 4.7 13.3 r 3.6 16.9 r 5.8 16.9 r 6.3 Cladosporium spp. 2.1 r 3.2 1.5 r 2.5 3.3 r 4.5 2.1 r 1.8 4.4 r 3.7 2.1 r 2.6 2.9 r 2.3 3.3 r 3.1 2.7 r 4.2 1.5 r 2.7 Curvularia spp. 4.0 r 4.3 2.7 r 4.1 5.2 r 5.0 2.9 r 3.3 3.1 r 3.0 1.0 r 1.7 0.0 r 0.0 0.0 r 0.0 0.8 r 1.6 1.0 r 2.0 Fusarium spp. 3.3 r 4.0 2.9 r 4.2 1.5 r 2.7 1.3 r 1.8 1.9 r 2.4 2.3 r 3.3 2.1 r 1.8 4.4 r 2.4 2.1 r 2.6 2.3 r 2.9 rr Mucor spp. 1.5 r 2.0 4.0 r 4.5 2.5 r 3.8 2.7 r 3.3 1.9 r 2.8 1.3 r 2.0 3.5 r 2.5 4.4 r 1.9 5.2 r 4.3 4.0 r 4.1 Penicillium spp. 2.7 r 2.9 6.7 r 5.3 4.0 r 3.9 6.3 r 6.1 4.4 r 4.1 4.8 r 4.3 5.8 r 1.9 3.1 r 2.6 2.7 r 3.9 9.4 r 5.8 Rhizopus spp. 2.5 r 4.1 2.9 r 3.8 1.9 r 2.4 2.5 r 3.5 1.3 r 2.1 2.5 r 2.8 0.8 r 1.6 2.7 r 2.3 1.9 r 2.8 1.7 r 2.5 Trichoderma spp. 1.5 r 2.5 2.1 r 2.3 0.8 r 2.5 2.7 r 4.2 1.9 r 3.0 1.0 r 2.0 0.0 r 0.0 0.0rr r 0.0 0.6 r 1.1 0.8 r 1.6 Others 5.6 r 3.4 6.5 r 3.3 5.6 r 3.6 6.3 r 2.7 6.3 r 2.5 2.9 r 2.6 6.3 r 2.7 8.8 r 1.3 3.3 r 3.7 4.8 r 3.3

Statistical Analysis: ANOVA; t-test; Mann-Whitney test Bus stand: Means of Aspergillus spp. in 2007 and in 2008 significantly (P<0.05) greater than means other species in the corresponding years; No significant (P>0.05) difference between the2007 and 2008 means of Aspergillus spp.

Railway station: Means of Aspergillus spp. in 2007 and in 2008 significantly (P<0.05) greater than means other species in the corresponding years; No significant (P>0.05) difference between the2007 and 2008 means of Aspergillus spp.

Recreation ground: Means of Aspergillus spp. in 2007 and in 2008 significantly (P<0.05) greater than means other species in the corresponding years; No significant (P>0.05) difference between the2007 and 2008 means of Aspergillus spp.

Sewage plant: Means of Aspergillus spp. in 2007 and in 2008 significantly (P<0.05) greater than means other species in the corresponding years; No significant (P>0.05) difference between the2007 and 2008 means of Aspergillus spp. Means of Alternaria spp., Fusarium spp. and Rhizopus spp. in 2007 and 2008 significantly (P<0.05) greater than other species.

Vegetable market: Means of Aspergillus spp. in 2007 and in 2008 significantly (P<0.05) greater than means other species in the corresponding years; No significant (P>0.05) difference between the2007 and 2008 means of Aspergillus spp.

Table 9. Total number and percentage distribution of bacteria (cfu/m3) in various microenvironments of indoor air during the study period (2007-2008)

Year 2007 2008 Microenvironment (n=2787.5; (n=2115; Total % 56.8%) 43.1%) (n=4902.5) Home 670 530 1200 24.5

Hotel 592.5 320 912.5 18.6

Office premise 580 485 1065 21.7

Public toilet 517.5 412.5 930 19

Theatre 427.5 367.5 795 16.2

Table 10. Distribution of bacteria (cfu/m3) in indoor air

Total number of bacteria (n=4902.5) 2007 2008 Month Office Public Office Public Home Hotel premise Toilet Theatre Total % Home Hotel premise toilet Theatre Total % January 65 40 47.5 60 47.5 260 9.3 52.5 27.5 42.5 37.5 30 190 9.0 February 60 55 52.5 42.5 25 235 8.4 42.5 27.5 42.5 32.5 37.5 182.5 8.6 March 57.5 57.5 62.5 42.5 35 255 9.1 37.5 42.5 47.5 47.5 37.5 212.5 10.0 April 65 75 55 47.5 30 272.5 9.8 37.5 37.5 37.5 55 20 187.5 8.8 May 35 40 45 45 52.5 217.5 7.8 30 30 32.5 40 27.5 160 7.6 June 67.5 45 30 27.5 45 215 7.7 52.5 7.5 42.5 20 27.5 150 7.1 July 35 40 40 45 37.5 197.5 7.1 40 20 37.5 35 35 167.5 7.9 August 70 42.5 52.5 40 25 230 8.2 50 35 55 27.5 27.5 195 9.2 September 60 45 52.5 42.5 22.5 222.5 8.0 40 35 45 47.5 37.5 205 9.7 October 62.5 50 45 42.5 30 230 8.3 60 30 30 22.5 15 157.5 7.4 November 62.5 45 45 42.5 52.5 247.5 8.9 47.5 7.5 42.5 27.5 35 160 7.6 December 30 57.5 52.5 40 25 205 7.4 40 20 30 20 37.5 147.5 7.0 Total 670 592.5 580 517.5 427.5 2787.5 99.9 530 320 485 412.5 367.5 2115 100

Table11. Type and total number of bacterial isolates (cfu/m3) from indoor air (2007 & 2008)

Indoor air (2007 & 2008) Organisms Office Public Home Hotel premise Toilet Theatre Total %

Staphylococcus spp. 387.5 250 267.5 190 207.5 1302.5 26.6

Micrococcus spp. 285 185 350 210 150 1180 24.1

Bacillus spp. 185 142.5 77.5 90 82.5 577.5 11.8

Aeromonas spp. 60 55 125 42.5 62.5 345 7.0

Escherichia coli 65 90 52.5 105 60 372.5 7.6

Pseudomonas spp. 17.5 47.5 35 40 27.5 167.5 3.4

Klebsiella spp. 20 30 5 35 12.5 102.5 2.1

Serratia spp. 0 0 0 12.5 0 12.5 0.2

Proteus spp. 40 10 22.5 40 35 147.5 3.0

Others 140 102.5 130 165 157.5 695 14.1

Total 1200 912.5 1065 930 795 4902.5 100

Table 12. Bacterial concentration (cfu/m3 Air; Mean r SD of 12 months) in different indoor locations during 2007 and 2008

House Hotel Office Public Toilet Theatre Species 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 Staphylococci spp. 19.0 ± 6.1 11.7 ± 6.2 12.5 ± 6.9 8.3 ± 5.4 9.4 ± 3.7 12.9 ± 4.0 8.5 ± 4.1 7.3 ± 5.9 8.5 ± 4.7 8.8 ± 3.1 Micrococcus spp. 13.3 ± 5.5 10.4 ± 4.7 9.2 ± 3.9 6.3 ± 4.6 17.9 ± 7.8 11.3 ± 5.2 6.9 ± 4.4 10.6 ± 3.4 4.8 ± 4.5 7.7 ± 4.8 Bacillus spp. 6.5 ± 4.2 2.3 ± 2.5 9.6 ± 5.9 2.3 ± 2.7 4.6 ± 3.5 1.9 ± 2.4 4.8 ± 3.4 2.7 ± 2.5 5.0 ± 3.2 1.9 ± 2.6 Aeromonas spp. 2.3 ± 2.3 2.7 ± 2.9 2.9 ± 2.8 1.7 ± 1.9 6.9 ± 4.9 3.5 ± 2.3 2.9 ± 2.6 0.6 ± 1.1 3.3 ± 2.7 1.9 ± 2.2 E.coli 2.9 ± 2.3 1.7 ± 1.9 5.4 ± 2.8 2.1 ± 1.8 2.3 ± 2.9 2.1 ± 2.1 5.8 ± 3.3 2.9 ± 3.0 2.9 ± 2.1 2.1 ± 2.3 Pseudomonas spp. 0.8 ± 1.2 0.6 ± 1.1 3.1 ± 2.4 0.8 ± 1.2 2.1 ± 2.6 0.8 ± 1.2 3.1 ± 3.7 0.2 ± 0.7 1.9 ± 1.9 0.4 ± 0.1 Klebsiella spp. 0.8 ± 1.6 0.4 ± 1.0 1.5 ± 2.0 1.0 ± 2.0 0.2 ± 0.7 0.2 ± 0.7 2.3 ± 2.0 0.6 ± 1.0 0.6 ± 1.1 0.4 ± 1.0 Serratia spp. ʊ ʊ ʊ ʊ ʊ ʊ 0.6 ± 1.1 0.4 ± 1.0 ʊ ʊ Proteus spp. 1.5 ± 1.7 1.9 ± 1.9 0.4 ± 1.0 0.4 ± 1.0 0.8 ± 1.6 1.0 ± 1.3 1.9 ± 2.2 1.5 ± 2.0 1.7 ± 1.9 1.3 ± 1.3 Others 5.4 ± 4.4 5.8 ± 3.7 4.8 ± 3.1 3.8 ± 3.8 4.2 ± 4.2 6.7 ± 3.4 6.3 ± 2.7 7.5 ± 2.8 6.9 ± 2.8 6.3 ± 2.7

Statistical Analysis: ANOVA; t-test; Mann-Whitney test Home: The concentration of Staphylococcus spp. in 2007 and 2008 significantly (P<0.05) different; greater than those of other species, in the respective years; The concentration of Micrococcus spp. significantly greater than those of other species other than Staphylococcus spp. in the respective years. Hotel: The concentration of Staphylococcus spp. in 2007 significantly (P<0.05) greater than those of other species; The concentrations of Micrococcus spp. and Bacillus spp. in 2007 significantly greater than those other species other than Staphylococcus spp. Office The concentration of Micrococcus spp. is significantly (P<0.05) greater than those of other species; The concentrations of premise: Staphylococcus spp. and Micrococcus spp. in 2008 significantly greater than those of other species in that year.

Public The concentration of Micrococcus spp. in 2008 is significantly (P<0.05) greater than those of other species. toilet: Theatre: The concentrations of Staphylococcus spp. in 2007 and 2008, and the concentration of Micrococcus spp. in 2008 significantly greater than those of other species in the respective years.

Table 13. Total number and percentage distribution of fungi (cfu/m3) in various microenvironments of indoor air during the study period (2007 – 2008)

Year 2007 2008 Microenvironment (n=2837.5; (n=2217.5; Total % 56.1%) 43.9%) (n=5055) Home 650 495 1145 22.7

Hotel 577.5 427.5 1005 19.9

Office premise 527.5 367.5 895 17.7

Public toilet 485 390 875 17.3

Theatre 597.5 537.5 1135 22.5

Table 14. Distribution of fungi (cfu/m3) in indoor air.

Total number of fungi (n=5055) 2007 2008

Month Public Office Public Home Hotel Office toilet Theatre Total % Home Hotel premise toilet Theatre Total % January 60 52.5 62.5 42.5 72.5 290 10.2 35 30 32.5 57.5 55 210 9.5

February 45 47.5 45 40 67.5 245 8.6 47.5 35 55 35 57.5 230 10.3

March 65 37.5 47.5 50 67.5 267.5 9.4 37.5 47.5 25 37.5 42.5 190 8.6

April 57.5 40 37.5 30 52.5 217.5 7.6 42.5 37.5 30 35 57.5 202.5 9.1

May 35 70 37.5 55 65 262.5 9.2 50 42.5 20 40 42.5 195 8.8

June 60 57.5 50 40 50 257.5 9.1 35 35 22.5 15 32.5 140 6.3

July 62.5 37.5 47.5 50 50 247.5 8.7 40 30 27.5 15 55 167.5 7.6

August 45 32.5 45 32.5 50 205 7.2 42.5 35 40 30 42.5 190 8.6

September 47.5 35 32.5 27.5 27.5 170 6.0 22.5 25 30 30 27.5 135 6.1

October 55 50 40 37.5 32.5 215 7.6 45 32.5 32.5 20 27.5 157.5 7.1

November 57.5 47.5 45 40 27.5 217.5 7.6 50 35 25 35 42.5 187.5 8.5

December 60 70 37.5 40 35 242.5 8.5 47.5 42.5 27.5 40 55 212.5 9.6

Total 650 577.5 527.5 485 597.5 2837.5 100 495 427.5 367.5 390 537.5 2217.5 100

Table 15. Type and total number of fungal isolates (cfu/m3) from indoor air (2007 & 2008)

Microenvironments (2007 & 2008) Organisms Office Home Hotel premise Public toilet Theatre Total %

Alternaria spp. 42.5 85 85 75 87.5 375 7.4

Aspergillus spp. 557.5 425 225 252.5 402.5 1862.5 36.8

Cladosporium spp. 87.5 57.5 85 75 80 385 7.6

Curvularia spp. 35 32.5 30 70 90 257.5 5.1

Fusarium spp. 27.5 52.5 87.5 42.5 82.5 292.5 5.8

Mucor spp. 77.5 60 62.5 90 62.5 352.5 7.0

Penicillium spp. 160 162.5 115 107.5 102.5 647.5 12.8

Rhizopus spp. 42.5 32.5 42.5 42.5 30 190 3.8

Trichoderma spp. 15 32.5 35 22.5 40 145 2.9

Others 100 65 127.5 97.5 157.5 547.5 10.8

Total 1145 1005 895 875 1135 5055 100

Table 16. Fungal concentration (cfu/m3 Air; Mean r SD of 12 months) in different indoor locations during 2007 and 2008

Home Hotel Office premise Public Toilet Theater Fungus species 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008

Alternaria spp. 1.5 r 3.6 2.1 r 4.4 5.4r 5.2r 1.7 r 2.2r 4.0 r 4.6 3.1 r 2.8 4.4 r 3.7 1.9 r 1.9 4.2 r 2.5 3.1 r 2.2 Aspergillus spp. 26.7 r rr7.5 19.8 r 9.2 21.7 r11.0 rrrrrrr 13.8 r 4.1 9.4 r 5.4 9.4 r 5.0 11.9 r 6.2 9.2 r 4.2 20.0 r 10.6 13.5 r 4.1 r Cladosporium spp. 5.6 r rrrrrrrrrr5.6 1.7 r 2.5 3.8 r 4.2 1.0 r 1.7 5.6 r 3.7 1.5 r 1.7 4.0 r 3.3 2.3 r 2.0 3.8 r 2.0 2.9 r 2.8 Curvularia spp. 2.5 r 4.9 0.4 r 1.4 1.7 r 3.1 1.0 r 2.0 2.1 r 2.6 0.4 r 1.0 2.7 r 3.8 3.1 r 3.7 5.6 r 3.4 1.9 r 1.9 Fusarium spp. 1.7 r 4.0 0.6 r 1.6 1.9 r 2.8 2.5 r 2.8 5.6 r 4.5 1.7 r 2.2 1.3 r 1.7 2.3 r 2.5 3.8 r 5.2 3.1 r 2.8 Mucor spp. 4.2 r 5.8 2.3 r 2.3 2.1 r 2.6 2.9 r 3.5 2.1 r 2.3 3.1 r 3.4 4.4 r 3.4 3.1 r 2.8 1.7 r 2.2 3.5 r 3.1 Penicillium spp. 4.2 r 5.1 9.2 r 5.5rrr 4.4 r 3.4 9.2 r 4.0 6.0 r 4.1 3.5 r 3.1 4.0 r 3.1 5.0 r 3.2 2.9 r 2.8 5.6 r 4.0 Rhizopus spp. 2.7 r 3.1 0.8 r 1.6 2.1 r 3.2 0.6 r 1.1 2.5r 3.0 1.0 r 1.7 1.5 r 2.3 2.1 r 2.3 0.8 r 1.6 1.7 r 1.9 Trichoderma spp. 0.8 r 1.6 0.4 r 1.0 1.9 r 2.4 0.8 r 1.9 1.7 r 2.2 1.3 r 1.7 1.0 r 2.0 0.8 r 1.6 1.7 r 1.9 1.7 r 2.5 Unidentified 4.4 r 4.0 3.7 r 4.3 3.3 r 3.3 2.1 r 3.3 5.0 r 3.8 5.6 r 4.7 5.4 r 4.2 2.7 r 2.5 5.4 r 5.8 7.7 r 3.3

All fungi 5.4 r rrr4.5 4.1 r 3.4 4.8 r 4.1 3.6 r 2.7 4.4 r 3.6 3.1 r 2.7 4.0 r3 .1 3.3 r 3.0 5.0 r 3.8 4.5 r 2.9

Statistical Analysis: ANOVA; F: 20.84; P=0.000; t-test; Mann-Whitney test Home The annual mean concentrations of Aspergillus spp. differ significantly (P<0.05); the mean concentration of 2007 being significantly (P<0.05) larger than that of 2008; the means of Aspergillus spp. significantly larger than those of other species; the 2008 mean of Penicillium spp. significantly (P<0.05) greater than that of 2007 and than those of other species, other than Aspergillus spp.

Hotel: Only in the cases of Alternaria spp., Aspergillus spp. and Penicillium spp., the respective means of 2007 and 2008 differ significantly (P<0.05). The mean concentration of Aspergillus spp. is significantly higher than those of other species, during 2007 and 2008 (P<0.05) . The mean of Penicillium spp. is significantly higher than those of other species, other than Aspergillus spp. (P<0.05).

Office premise: Means of Aspergillus spp. significantly (P<0.05) higher than the means of other of the respective years; 2007 mean of Cladosporium spp. significantly (P<0.05) greater than its 2008 mean.

Public toilet: Means of Aspergillus spp. of 2007 and 2008 differ significantly (P<0.05); Means of Aspergillus spp. differ significantly (P<0.05) from the means of other species .

Theater: Means of Aspergillus spp. of 2007 and 2008 differ significantly (P<0.05); Means of Aspergillus spp. differ significantly (P<0.05) from the means of other species.

Table 17. Average monthly mean temperature (° C), monthly mean relative humidity (%), monthly mean rainfall (mm) and wind speed (km/h) of the locality in 2 sampling years.

Temperature (°C) Relative humidity (%) Rainfall (mm) Wind speed (Km/h) Month 2007 2008 2007 2008 2007 2008 2007 2008 January 30.4 29.7 77 80 0 50.2 7 6 February 31.2 31.3 74 80 6.6 10 5 5 March 32.9 32.2 77 81 0 137.9 5 4 April 34.6 34.5 76 73 0.2 26.4 6 5 May 39.2 39.5 62 57 0.1 0.3 7 7 June 35.8 37.1 71 63 94.2 126.4 7 7 July 35.3 36.1 73 66 243.9 28.6 7 7 August 34 34.7 79 73 170.9 147.2 7 7 September 34.2 34.5 73 72 167.7 120.9 6 6 October 32.7 32.4 79 85 274.9 372.9 6 5 November 30.9 31 80 81 95 556.6 6 7 December 29.5 30.2 85 82 253.3 17.7 7 6

Table18. Total number and percentage distribution of Aspergillus from various microenvironments during 2007 and 2008

Outdoor Indoor Organisms Total Total BS RS RG SP VM HM HT OP PT TH

Total fungi 1045 985 842.5 1000 972.5 4845 1145 1005 895 875 1135 5055

Aspergillus spp. 315 317.5 262.5 300 405 1600 557.5 425 225 252.5 402.5 1862.5

Percentage 30.10 32.20 31.10 30 41.60 33 48.60 42.30 25.10 28.80 35.40 36.80

Table 19 a). Monthly concentrations (cfu/m3 Air) of Aspergillus during 2007 and 2008

Month 2007 2008

January 17.5 ± 9.0 13.5 ± 5.0 February 17.8 ± 8.9 17.5 ± 5.1 March 15.6 ± 10.3 16.5 ± 5.4 April 15.8 ± 8.5 15.3 ± 6.9 May 17.5 ± 10.2 11.5 ± 5.0 June 18.3 ± 8.4 12.5 ± 7.5 July 15.8 ± 6.3 11.8 ± 7.8 August 15.0 ± 10.7 13.5 ± 7.7 September 5.8 ± 5.0 11.5 ± 5.4 October 18.3 ± 6.1 12.3 ± 6.8 November 12.8 ± 9.2 12.3 ± 3.2 December 17.8 ± 10.2 13.3 ± 7.6

b). Annual concentrations (cfu/m3 Air; Mean r SD of 12 months) of Aspergillus in different outdoor and indoor locations during 2007 and 2008

Sampling Locations t-test 2007 2008 Outdoor T P Bus stand 12.9 ± 6.6 13.3 ± 5.4 -0.17 0.866 Railway station 11.7 ± 5.4 14.8 ± 7.8 -1.14 0.267 Recreation ground 11.5 ± 8.4 10.4 ± 4.1 0.39 0.703 Sewage treatment plant 11.7 ± 4.9 13.3 ± 3.6 -0.95 0.355 Vegetable market 16.9 ± 5.8 16.9 ± 6.3 0.00 1.000 Indoor Home 26.7 ± 7.5 19.8 ± 9.2 2.01 0.050** Hotel 21.7 ± 11.0 13.3 ± 4.1 2.34 0.036* Office premise 9.4 ± 5.4 9.4 ± 5.0 0.00 1.000 Public toilet 11.9 ± 6.2 9.2 ± 4.2 1.25 0.226 Theatre 20.0 ± 10.6 13.3 ± 4.1 1.98 0.068 * Significant (P<0.05) Statistical Analysis: ANOVA; t-test; Mann –Whitney test.

Table 20. Characteristics of the study population

Controls Cases Characteristics Number % Number % Total 25 115 Gender Male 11 44 46 40 Female 14 56 69 60

Parental atopy and / or asthma 2 8 5 4.3 ETS1 In the Workplace 4 16 11 9.6 In the Home 2 8 5 4.3 Any work exposure2 6 24 22 19.1

ETS’: Environmental Tobacco Smoke

Exposure to sensitizers (Dusts, except moulds)

Table 21. Percentage and sex wise distribution of Case – Control study population

Age group (in years) Gender 21 - 30 % 31 – 40 % 41 - 50 % 51 – 60 % > 60 % Case study

Male 15 13 13 11 13 11 4 4 1 1 Female 22 19 24 21 12 10 6 5 5 4

Control study

Male 3 12 2 8 3 12 2 8 1 4 Female 3 12 4 16 5 20 2 8 0 0

Table 22. Percentage of Eosinophils in subject study

Subjects Controls Eosinophil % (n=115) (n=25) 1 83 12 2 1 7 3 12 4 4 5 2 5 5 0 6 7 0 8 2 0

Table 23. Total serum IgE levels (< 160 IU/ml; Mean ± SD) in different age groups of persons showing allergic symptoms and persons with no allergic symptoms

Age Groups Persons showing 21-30 31-40 41-50 51-60 Above 60 Male Female Male Female Male Female Male Female Male Female

82.7 ± 3 Allergic symptoms 90.4 ± 42.6 79.0 ± 38.6 89.0 ± 38.9 73.9 ± 29.4 77.8 ± 41.1 48.6 ± 16.3 56.9 ± 14.8 71.0 44.8 ± 10.4 4.6 (17s) (12) (20) (11) (9) (3) (5) (1) (4) (94) (12)

No Allergic symptoms 91.2 ± 38.8 101.9 ± 64.2 86.7 ± 7.6 91.8 ± 30.0 91.4 ± 23.9 56.4 ± 16.5 50.1 ± 2.7 74.1 ± 7.1 92.1 — (25) (3) (3) (2) (4) (3) (5) (2) (2) (1)

Note: Values shown in parentheses, n

Statistical Analysis: ANOVA; F: 1.107; P: 0.36; No significant difference between the means of the different groups

Table 24. Frequency of different age-groups of persons showing allergic symptoms

and having total serum IgE levels above 160 IU/ml

Serum total IgE level (IU/ml) Age Groups 161-320 321-480 481-640 641-800 801-960 M F M F M F M F M F 21-30 1 0 1 2 0 1 0 2 1 0 31-40 0 2 0 0 1 1 0 0 0 1 41-50 1 0 1 1 0 0 0 1 0 1 51-60 0 0 1 0 0 0 0 1 0 0 61 & above 0 1 0 0 0 0 0 0 0 0

Table 25. Frequency of different age-groups of persons having

Serum Specific IgE levels (IU/ml)

Serum specific IgE level (IU/ml) Age Groups 161-320 321-480 481-640 641-800 801-960 M F M F M F M F M F 21-30 1 0 1 2 0 1a 0 1+1c 1 0 31-40 0 2 0 0 1b 1 0 0 0 1 41-50 1 0 1 1 0 0 0 1 0 1 51-60 0 0 1 0 0 0 0 1 0 0 61 & above 0 1 0 0 0 0 0 0 0 0

a serum contained the specific IgE (512.1 IU/ml) bserum contained the specific IgE (482.6 IU/ml) c serum contained the specific IgE (706.8 IU/ml)

Fig.2. Air sampling in outdoor microenvironments

a) Bus stand b) Railway station

c) Recreation ground

d) Sewage treatment plant e) Vegetable market

  









Fig.3. a) Exposed plate showing bacterial colonies on Nutrient agar

b) Exposed plate showing bacterial colonies on Blood agar





















Fig. 4. Bacteria isolated from various microenvironments

Staphylococcus sp. Micrococcus sp.

Aeromonas sp. Bacillus sp.

E.coli Klebisella sp.

Proteus sp. Pseudomonas sp.

Fig.5 (a – j). Month wise percentage distribution of bacteria Percentage distribution Percentage distribution

Fig.5. Contd… Percentage distribution Percentage distribution

Fig.5. Contd… Percentage distribution Percentage distribution

Fig.5. Contd… Percentage distribution Percentage distribution

Fig.5. Contd.... Percentage distribution Percentage distribution

Fig. 6. Percentage distribution of bacteria in various outdoor microenvironments

Fig.7 (a – e). Annual loads (Mean ± SD) of different bacterial species in an outdoor environment

Fig.7. Contd...

Fig.7. Contd....

Fig.7. Contd...

Fig.7. Contd...

Fig.8. a) Exposed plate showing fungal colonies from an outdoor microenvironment

b) Exposed plate showing fungal colonies from an indoor microenvironment

Fig. 9. Fungi isolated from microenvironments

Alternaria sp Aspergillus sp

Fusarium sp. Curvularia sp.

Penicillum sp Cladosporium sp

Trichoderma sp Rhizopus sp

Fig. 10 (a – j). Prevalence and month wise percentage distribution of fungi from Outdoor microenvironment Percentage distribution Percentage distribution

Fig.10. Contd… Percentage distribution Percentage distribution

Fig.10. Contd… Percentage distribution Percentage distribution

Fig.10. Contd… Percentage distribution Percentage distribution

Fig.10. Contd… Percentage distribution Percentage distribution

Fig.11. Percentage distribution of outdoor fungi from various microenvironments

Fig. 12(a – e). Annual loads (Mean ± SD) of different fungal species in an outdoor environment

Fig.12. Contd…

Fig.12. Contd…

Fig.12. Contd…

Fig.12. Contd…

 

Fig. 13. Air sampling in indoor microenvironments

a) Home a)b) HomeHotel b) Hotel

‘c) Office premise

d) Public toilet e) Theatre

Fig.14 (a – j). Month wise percentage distribution of bacteria from an indoor environment Percentage distribution Percentage distribution

Fig.14.Contd... / Percentage distribution Percentage distribution

Fig.14.Contd... / Percentage distribution

Fig.14.Contd... / Percentage distribution

Fig.14.Contd... / Percentage distribution Percentage distribution

Fig.14.Contd... / Percentage distribution Percentage distribution

Fig.15. Percentage distributions of bacteria in various indoor microenvironments

Fig.16 (a – e). Annual loads (Mean ± SD) of different bacterial species in an indoor environment

Fig.16.Contd..

Fig.16.Contd..

Fig.16.Contd..

Fig.16.Contd..

Fig. 17 (a – j). Prevalence and month wise percentage of fungi from an indoor microenvironment Percentage distribution

 Percentage distribution











Fig 17. Contd… Percentage distribution

 Percentage distribution











Fig 17. Contd… Percentage distribution

 Percentage distribution

 







Fig. 17 Contd.. Percentage distribution Percentage distribution

Fig 17. Contd… Percentage distribution

 Percentage distribution

Fig.18. Percentage distributions of indoor fungi from various microenvironments

Fig. 19 (a – e) Annual loads (Mean ± SD) of different fungal species in an indoor environment







Fig 19 Contd…









Fig19. Contd…





Fig 19 Contd…





Fig 19 Contd…



Fig.21. Monthly concentration of Aspergillus (Mean ± SD) from microenvironments





Fig. 22. Age wise distribution of study population







Fig.23. a) Percentage frequency of Serum IgE levels in all age groups

/

b) Total Serum IgE (<160 IU / ml)

Fig.24. a) Immuno assay method for total serum IgE level

b) Microtitre wells showing positive reactions

Fig. 25. Total serum specific IgE level (IU /ml)

Fig.26. a) Air sanitizer (Bacillocid)

b) Comparison of fungal colonies before and after sanitization

5. DISCUSSION

It is essential to evaluate the quality of air we breathe whether indoors or outdoors. The number and type of air borne microorganisms can indicate the degree of cleanliness and may be a source of human discomfort. The present study examined the bioaerosol level of five different microenvironments each from outdoor and indoor air. The major parameters associated with the bioaerosol measurements included the microenvironment type, sampling time and seasonal distribution. No major environmental problems were reported at the microenvironment investigated during the entire survey period.

5.1. Outdoor microbial concentration

5.1.1. Bacteria

Regardless of the season, bacteria were detected (total counts in cfu/m3) in all the outdoor air samples. However, the occurrence of individual bacterial species and the outdoor bacterial concentrations (5495 cfu/m3) (Table 1) and their seasonal distributions were significantly higher than the outdoor total fungal concentrations (4845 cfu/m3) (Table 5). This is also supported by Pastuszka et al. (2000) with an outdoor total bacterial count (4344 cfu/m3) significantly higher than the outdoor total fungal count (4121 cfu/m3) from an outdoor environment in Upper Silesia. One possible cause is that the soil surface would be a significant source of bacteria, since higher concentrations of bacteria were present when dust was raised (Jones and Harrison, 2004). Moreover, the variation of outdoor bacterial concentrations according to atmospheric height is closely related to local meteorological parameters such as turbulence and mixing height (Hirst et al., 1967). Mandrioli et al. (1983), who measured bioaerosol concentration at various heights from the ground to 6000m, reported a similar decreasing trend with height in bioaerosol concentrations. However, Mandrioli et al. (1983) also found that, on another day under a different meteorological condition, a profile of culturable bacteria concentrations with height showed little variations.

The outdoor exposure to bioaerosols can be obtained from the detailed analysis of the bacterial genera. Staphylococcus spp. was the predominant bacterial type in almost all the outdoor air studied and had the highest count; constituting 21.1% (Table 3) of the total bacterial genera whereas the second and the third predominant groups were Bacillus, constituting 18.5% and Micrococcus of 17.2% of total respectively. Other bacterial genera were also present at a

52 lower frequency. The outdoor bacterial concentration of Bacillus spp. in 2007 was significantly (Mean ± SD 11.5 ±2.7 cfu/m3) (Table 4) higher than those of other species (P< 0.05) from the microenvironment of Bus stand. Concentrations of Staphylococcus and Micrococcus were significantly (P< 0.05) greater than 2008 than those of other species in that year. In general, these two bacterial groups were significantly higher in microenvironments of railway station, recreation ground and vegetable market. On the other hand E.coli with a concentration of 12.9± 7.0 cfu/m3 (Mean ± SD; 2007) and 11.5± 6.8 cfu/m3 (Mean ± SD; 2008) were significantly higher (P< 0.05) than those of other species from sewage treatment plant. The present study reveals the presence of higher total bacterial concentrations from outdoor environment which is comparable to those in other reports with mean bacterial values between 10 and 103cfu/m3 (Jo and Seo, 2005).

The last decade has a significant increase in scientific data on non-occupational exposure as well as occupational exposure to bioaerosols in many developed countries for the purpose of evaluating the relationship between exposure and health effects (Gorny and Dutkiewicz, 2002). However, there is only limited amount of information currently available for Chennai on individual exposure to bioaerosols, including a few reports on certain public access facilities such as hospitals. Thus it is reasonable that such recreation grounds, bus stands and railway stations with a high human occupancy should be investigated as regards the exposure of the individuals to bioaerosols, and the results used to evaluate the relationship between exposure and health effects. Children spend a large portion of their week end time in recreation ground and are also considered as potentially more vulnerable than adults, and their health is more susceptible to environmental exposure (Guzelian et al., 1992; Aprea et al., 2000).

Any reports on the prevalence of such microbe in outdoor environment in major cities are of importance in providing protection to the population of the city. Most bacteria or bacterial agents are not very potent allergens with the exception of the spore forming Actinomycetes. Bacterial cell wall components, such as endotoxin of Gram negative bacteria and peptidoglycan of Gram positive bacteria are agents with important pro inflammatory properties that may induce respiratory symptoms. The toxigenic and pathogenic potential of these microbes have been well documented in literature (Rylander and Jacob, (1997). The microbial load which is commonly found has been implicated in causing primary and secondary infections in susceptible individuals. To minimize the exposure, spitting in public places to be banned and water stagnation to be drained.

53

Bacteria from Enterobacteriaceae in high quantities were detected in the air at the aeration tank, during the operation. The presence of pathogenic bacteria in aerosols does not always cause pathogenic alterations. Induction of the latter is directly related to the quantity of microorganisms and an organism vulnerability to infection. It is reported that Sewage Treatment Plant (STP) employees gain resistance to sewage aerosols. On the other hand, the presence of pathogenic microorganisms can be especially risky to persons accidently present in a STP area (Filipkowsk et al., 2002). Sewage treatment plant facilities have been found to generate bioaerosols, which are transported by the prevailing winds down streams to areas that can be up to several hundred meters away. STP represents an important source of bioaerosol emission, especially the stages that include moving parts or sudden drop of over flowing liquids resulting in bioaerosols formation (Sawyer et al., 1993; Brandi et al., 2000; Ranali et al., 2000; Bauer et al., 2002; Pascual et al., 2003). Droplets produced might contain therefore, varying amounts of pathogenic microorganisms, some of them with the ability to infect a person through the respiratory system, contact or swallowing. Studies show a significant connection between bioaerosols and cases of respiratory and intestinal diseases (Sawyer et al., 1993).

The present study estimated the presence and concentration of bioaerosols in the air surrounding installations of sewage treatment plants especially E.coli and Klebsiella spp. as being indicators for intestinal infection. Many other sources of microorganisms containing aerosols are, however, generated through human activities in both urban and rural areas. Population growth in urban areas increased the density of domestic wastes which must be disposed off in a safe and environmentally sound manner. Consequently, expansions of existing waste treatment in utilization facilities are necessary. Some of these facilities have, however, been shown to emit microorganisms containing aerosols under certain conditions. Sewage treatment plants have been considered as potential sources of air borne infectious microorganisms (Sawyer et al., 1993). Because of economic, environmental or political constraints, some of these facilities are located in densely populated regions of urban or suburban communities. In these cases, a determination of the contribution of the facilities to the microorganism’s content of the ambient air may allow an elevation of the potential for adverse health or environmental effect.

The air near residential environments is not sterile but contains microorganisms, some of potential enteric origin. This study confirms, in the case of small treatment plants, the presence of airborne bacteria from aerated sewage. It is particularly note worthy that enteric bacteria were isolated frequently from upwind samples taken close to aerated sludge tanks. Aerosol samples

54 collected at least 10 km from any known sources of aerated sewage seldom yielded enteric bacteria. In a study of coliforms emitted from effluents sprays, Teltsch and Katznelson (1978) reported the effects of variation from other factors. Under these conditions, there was a positive correlation between bacterial counts and relative humidity and negative correlation with solar irradiation.

The hazards associated with exposure to airborne enteric microorganisms are not known but, based on available evidence the risk cannot be disregarded. This study indicates that persons residing or working close to such sewage plants may have an increased probability of contact with enteric organisms. As already pointed out, Sewage treatment facilities have been found to generate bioaerosols which are transported by the winds downstream to areas that can be up to several 100 meters away. Number of aerosols is slowly added to the environment through various means. The load may be suddenly increased due to various treatment processes. People working in such sewage treatment plants can prove to be a hazardous occupation, with illness deriving from water infection, representing the basic point of interest in the study of workers health. The cause of bioaerosol can be controlled by maintaining proper and hygienic environment.

5.1.2. Fungi

Any atmospheric air contains spores of certain fungal species. Depending on the living conditions, environmental and climatic conditions, fungal concentrations in outdoor air can vary greatly. The present investigation recorded a total fungal count of 4845 cfu/m3 (Table. 5) during the study period from various microenvironments.

In Chennai, the fungal concentrations in outdoor samples (Table. 5) were less, compared to those of some other world cities (Fang et al., 2005; Solomon et al., 2006). For example, Fang et al. (2005) reported a concentration ranging from 24 to 13,960 cfu/m3 in the city of Beijing, China. Solomon et al. (2006) estimated a very high mould spore concentration ranging from 21,000 to 102,000 spores/m3 of outdoor air in New Orleans, Louisiana, USA. In European hospital (Italy), the maximum fungal concentration recovered in outdoor air was 3150 cfu/m3 (Pini et al., 2004), almost six times more than the maximal value observed at the Dijon site (Sautour et al., 2009). These differences could be explained by different sampling methods used to measure fungal concentrations, air flow rate, duration of sampling and culture medium. However, the present results were consistent with those reported in Paris (Dassonville et al., 2008).

55

Concerning the percentage of fungi isolated in outdoor air during the study period (Table .7), it was noticed that many of the main fungal genera that recovered were similar to those observed in some other cities. Aspergillus was the most prominent fungus and was reported almost from all samples. The percentage of Aspergillus was 33 in Chennai which supported the other results (Sautour et al., 2009). Penicillium was always the most frequently identified genus with the percentage ranging from 10 to 28 in other cities (12.3% in Chennai) (Sautour et al., 2009), Alternaria from 4 to 14% in other cities (7.7% in Chennai) (Pini et al., 2004; Fang et al., 2005; Gomez de Ana et al., 2006; Lee and Jo, 2006) and inturn Cladosporium ranging from 43 to 78%, greatly higher than the present result (6.4% in Chennai). Other fungal genera were also present at a lower frequency.

The present study reveals that the outdoor fungal concentration (Mean±SD) of Aspergillus in 2007 and 2008 was significantly (P < 0.05) greater than those of other species in the corresponding years from the microenvironments of the study areas. No significant (P > 0.05) difference between 2007 and 2008 in means of Aspergillus in all the outdoor microenvironments. Aspergillus seems to be the predominant fungus followed by Penicillium (Mean ±SD) in 2007 and 2008 was significantly (P<0.05) greater than those of other species in the corresponding years from the microenvironments of bus stand, sewage treatment plant and vegetable market. Among the remaining fungal groups, genera such as Alternaria (3.5±2.3; 2007 and 6.7± 3.3; 2008), Fusarium (2.1 ± 1.8; 2007 and 4.4 ± 2.4; 2008) and Rhizopus (0.8± 1.6; 2007 and 2.7± 2.3; 2008) were significantly greater over other groups of fungi in sewage treatment plant (P< 0.05) (Table.8).

Just as the present study, Ren et al. (1999) showed that Aspergillus, Penicillium, Cladosporium and Alternaria are the predominant genera in outdoor air (Table.7). Ebner et al. (1989) found species of Aspergillus in high concentrations especially in late fall and following rainy weather.

According to Reiss (1998), thin walled colorless spores such as from Aspergillus are quickly eliminated by the ultraviolet radiation in the sun light. Possibly the absence of significant sources in summer and the extremes of temperature and humidity besides the fungicidal effect of ultraviolet might explain why species of Aspergillus and Penicillium. were found more frequently in outdoor in spring, fall and winter. Generally, the spores of Penicillium spp. and Alternaria spp. were found in relatively low concentrations. Previous studies report

56 comparatively on the concentrations of fungal species in outdoor air (Li et al., 1995; Millington and Corden, 2005).

In addition, Hyvarinen et al. (1993) and Niemeier et al. (2006) reported that more fungal species were identified by air sampling than by swab sampling. All of these methods complement each other and may be useful in specific cases. The species of Penicillium have been identified as important causative agents of extrinsic bronchial asthma (Shen and Han, 1998). It has been reported that most common genera namely Aspergillus, Penicillium, Cladosporium and Alternaria should always be considered as a cause of fungal allergy (Peat et al., 1993). In the present study, although some degrees of seasonal variations of the major genera were detected, the most notable ones were the Aspergillus and Penicillium.

Many sources of bioaerosols are man – made such as sewage treatment and vegetable waste disposal facilities and so on. Dust levels in an open air are relatively high, especially when the outdoor activity is more. Higher bioaerosol levels occur when dust is stirred up during outdoor cleaning, playing and dumping waste materials. Similarly, these cause a lot of human health problem. The amount of refuse collected from urban areas in India ranges in the order of 0.3 to 0.5 kg/person per day including night soil (Rao, 1995). Microorganisms are introduced into the atmosphere from these sources, transmitted via the air stream, and finally get deposited on some surface (Lighthart and Frisch, 1976). Particles may be carried by air currents to some distance, their source and may present occupational as well as public health considerations.

Concentrations at most sampling locations indicated atmospheric dispersion from the facility. However bioaerosol levels at some locations deviated from the pattern. Several colonies of Bacillus, soil bound microorganisms were identified on outdoor samples only and apparently was the primary cause of high concentrations upwind of the building.

The organic sources for proliferation of moulds were ample in the environments. This is same in case of vegetable markets. There are many reports available on the level of mesophilic fungi present in the atmosphere of occupational environments (Udaya Prakash and Vittal, 2005). Accumulations of waste vegetables are thrown in and around the market which favour the growth of microbes in vegetable market. The humidity generated while rotting of vegetables and heats generated due to piling of decaying vegetables provide a cocktail for these organisms to grow and proliferate. It was reported that moist, sun heated piles of plant substrates favour the growth of these fungi (Mouchacca, 1995).

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5.2. Indoor microbial concentration

5.2.1. Bacteria

The present study shows the total indoor bacterial concentration (4902.5 cfu/m3) (Table.9) slightly less from the indoor fungal concentration (5055 cfu/m3) (Table.13). However the indoor bacterial concentrations were significantly higher in few microenvironments than the indoor fungi and outdoor bacterial concentrations. Similarly Scheff et al.(2000) reported that in a middle school of Spring field, Illinois, which had no known environmental problems, for total bacteria, the indoor concentrations (AM: 561 cfu/m3) were significantly higher than the outdoor concentrations (AM: 389 cfu/m3). The present study also showed a significant higher indoor bacterial concentration of 1200 cfu/m3 from a microenvironment of home (Table.9). The same result was reported by Pastuszka et al. (2000) in healthy homes of Upper Silesia, Poland and Hargreaves et al. (2003) reported for homes in Brisbane, Australia. In contrast to this report, the present study revealed a lower indoor bacterial concentration from the microenvironments of office premises, public toilets and theatres than the outdoors. The bioaerosol concentrations in Chennai are similar to those from other reports, with bacterial values between 10 and 103 cfu/m3 (Nevalainen, 1989; Flannigan, 1993; Gall up et al., 1993; Ren et al., 1993; Dekoster and Thorne, 1995; Seltzer, 1995). Reports are also available on the bacterial levels of residences, which varied between 10 and 104 cfu/m3 (Macher et al., 1991; Nevalainen et al., 1991; Reponen et al., 1992; Dekoster and Thorne, 1995; Rautiala et al., 1996; Ross et al., 2000; Pessi et al., 2002).

In this study, the highest concentration of airborne bacteria measured in the office buildings was 580 and 485 cfu/m3during 2007 and 2008 respectively (Table.9). Most of these buildings were equipped with a modern ventilation system. This result differs from an annual concentration level of bacterial aerosol in a microenvironment of office premise from Upper Sielsia (Pastuszka et al., 2000) with a total bacterial count of 956 cfu/m3 and was more than sabout 1.5 times higher than the present work. In the office environment, the concentration of air borne bacteria was approximately 1065 cfu/m3 (2 year study) which was slightly less than in home environment (1200 cfu/m3). The reason may be more sources of bacteria in homes (for example dogs, cats and other animals). The difference can also be the result of conditions less conducive to bacterial growth in offices than in homes as typically in contrast to homes, there are no carpets in Chennai offices and in most of the floor is wet cleaned daily. Similar concentration levels of bacterial aerosols were observed from hotel and in home environments. In case of public toilet, the risk of illness or infection has been linked to faecal contamination of

58 the water, due to faeces released or introduced into the water when a person has an accidental faecal release is also a potential source of pathogenic organisms. In addition, infected users can directly contaminate and opportunistic pathogens (mainly bacteria) can also be shed from users and transmitted via aerosols. In addition, facilities (ventilation, air conditioning system, proper disposal) or on other wet surfaces within the facility to a point at which some of them may cause a variety of infections such as bacterial diarrhoea, throat infection, fungal skin infections and so on. Proper guidance and self hygiene is most important for any person availing public facilities.

Important information about the indoor exposure to bioaerosols in Chennai can be obtained from the detailed analysis of the bacterial genera. Species of Staphylococcus were present in almost all the indoor air studied; these bacteria also had the highest count, constituting 26.6% of the total bacterial genera (Table.11). The second most common bacterial aerosol was Micrococcus spp. constituting 24.1% of total. The present results differ from the indoor exposure to bioaerosols in Upper Silesia where species of Micrococcus were present in all homes studied and have the highest count, constituting 36% of the total bacterial genera and the second most common bacterial aerosol was Staphylococcus epidermidis present in 76% of home studied constituting 14% of the total. However species of Staphylococcus were the most frequently occurring indoor bacteria in Chennai followed by Micrococcus which contributed both together about 50% of the total bacteria concentration. The present results support the general statement that the bacteria in the indoor air are dominated by species of Staphylococcus and Micrococcus likely to be the most prominent (CEC, 1993; Gall up et al., 1993; Maroni et al., 1993). Other bacterial genera were present at a lower frequency. Most people are exposed to contaminated indoor environments regardless of its climatization situation (Sorenson, 1987; Rylander et al., 1989; Burge, 1990; Gravesen et al., 1990).

The present study also reveals, the indoor bacterial concentration (Mean± SD) of species of Staphylococcus in 2007 and 2008 significantly (19.0 ± 6.1 and 11.7± 6.2 cfu/m3) (Table.12), different and greater than those of other species in the respective years (P< 0.05). The concentration of species of Micrococcus was significantly greater than those of other species other than Staphylococcus from the microenvironment of home. Similarly, the concentration of Staphylococcus (Mean± SD) was significantly greater (12.5± 6.9; 2007 and 8.3± 5.4; 2008), (Table.12), than those of other species (P<0.05) in hotel environment where as in office premise and public toilets, species of Micrococcus were significantly greater (P< 0.05) with 17.9±7.8; 11.3±5.2 during 2007 and 2008 respectively. Theatre environment also showed with the

59 concentration of species of Staphylococcus in 2007 and 2008, and species of Micrococcus in 2008 significantly greater in the respective years.

5.2.2. Fungi

While occupational exposure to air borne pollutants such as asbestos and coal dust is known to cause lung cancer and Pneumoconiosis (black lung disease), consequences of air contaminants especially bioaerosols, in homes and non industrial work sites such as office buildings are not yet fully understood. In the 1970’s and 1980’s microbial contamination was identified as the primary cause for poor air quality in only 5% of more than 500 indoor air quality (IAQ) investigations conducted by National Institute for Occupational Safety and Health (NIOSH); while the remaining 95% resulted from inadequate ventilation, entrainment of outdoor air contaminants, contaminants in building fabric and unknown sources (NIOSH, 1989). However, in the last 10 years microorganisms were the primary source of indoor air contamination in as many as 35 – 50% of IAQ cases (Lewis, 1994). This change has been attributed atleast partially to a paradigm shift from chemical contaminant based investigations to an interdisciplinary approach combining evaluation of physical, chemical and microbiological constituents of indoor air environments. As regards the type of microenvironment, indoor total bacterial count was lower than the total fungal count. One possible cause for this difference was the higher occupancy and activity in indoor was found to be closely related indoor microbial levels (Scheff et al., 2000), while settled spores were resuspended in indoor air by air movement caused by human activities such as walking and running (Buttner and Stetzenbach, 1993).

Fungi are ubiquitous organisms that make up approximately 25% of earth’s biomass. Moulds are very adaptable and can colonize dead and decaying organic matter (Wood, Paper, leather, textiles) and even damp, inorganic material (glass, painted surfaces, bare concrete) if organic nutrients such as dust or soil particles are available. Because various genera grow and reproduce at different substrates, water concentrations and temperatures, moulds occur in a wide range of habitats (Sandra et al., 2003).

Mould types and concentrations of indoors are primarily a function of outdoor fungi and substrate water (related to indoor humidity level). Most indoor moulds originate from exterior sources; some species of Aspergillus and Penicillium can grow and reproduce effectively indoors and are commonly found in air samples of normal “dry” buildings (Sandra et al., 2003). Moulds are composed of linear chains of cells (hyphae) that branch and intertwine to form the fungus body (mycelium). All fungal cell walls contain (1- 3) - ȕ – D – glucane, a medically significant

60 glucose polymer that has immunosuppressive, mitogenic and inflammatory properties. This mould cell wall component also appears to act synergistically with bacterial endotoxins to produce airway inflammation following inhalation exposure (Fogelmark et al., 1994).

Moulds are important potential producers of toxins of indoors that can contain species of Aspergillus, Penicillium and Fusarium (Beasley, 1994). Two classes of mycotoxins have been isolated from house dust samples: aflotoxins from some strains of Aspergillus flavus and trichothecenes from some species and strains of Fusarium, Cephalosporium, Stachybotrys and Trichoderma. Several reports have associated over growths of trichothecene producing fungi with human health effects such as cold and flu like symptoms, head ache and general malaise (Croft et al., 1986; Johanning et al., 1993; Nikulin et al., 1994). However, isolation of a toxogenic fungus from a building does not imply the presence of mycotoxin, since the physical conditions necessary for mycotoxin production are very specific, and are often different from those required for growth of parent mould. Likewise, failure to produce toxins invitro does not mean that a mould known to be toxogenic will not produce toxins in a field situation (Beasley, 1994). Moulds also produce a large number of volatile organic compounds (VOCs). These chemicals are responsible for the musty odors produced by growing moulds. There is little evidence that fungal VOCs cause specific human health effects (Batterman, 1995) but the most common VOC, ethanol is a potent synergizer of many fungal toxins.

A strong relation was found between outdoor and indoor airborne fungi. Many authors also reported that outdoor air is the major source of indoor fungi (Li et al., 1995; Ren et al., 1999; Su et al., 2001; Chew et al., 2003; O’ Connor et al., 2004; Lee and Jo, 2006). Supporting to the present work, indoor Aspergillus and Penicillium levels tended to be higher due to potential indoor sources (Table.15). No association was noted with these fungal concentrations probably due to the fact that these fungi had indoor sources. In a previous study by Ren et al. (2001), the presence of air conditioning was associated with a decrease in total air borne fungal levels, perhaps due to a lack of window opening. In other studies, it has been reported that total airborne fungal genera and their concentrations were lower in winter than in other seasons (Ren et al., 1999, 2001; Chew et al., 2003; Lee and Jo, 2006) but the present study revealed differently and correlates with a study reported in southern Taiwan (Pei – Chih et al., 2000; Su et al., 2001) with a higher level of indoor fungi obtained during the month of January (winter), (Table .14).

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Concerning percentages of fungi isolated in indoor air during the study period, it was noticed that many of the main fungal genera that recovered were similar to those that observed in some other cities. Aspergillus was the most predominant fungus and was reported almost from all the indoor samples and its distribution revealed 36.8% (Table 15) which is higher than the outdoor percentage (33%) (Table. 7). Penicillium spp. was always the most frequently identified genus with the percentage of 12.8 in Chennai which supports in other cities (Li and Kendrick, 1995; Lee and Jo, 2006). It is followed by Cladosporium (7.6%) and Alternaria (7.4%). Other fungal genera were also present at a lower frequency (Table 15). Concerning percentages of fungi isolated in indoor air, species of Aspergillus and Penicillium were by far the most frequently encountered genera. The prevalence of these two genera has been previously observed in Dijon hospital (Sautour et al., 2007), other hospitals (Wu et al., 2000), and recently in new born babies’ homes (Paris, France) (Dassonville et al., 2008). This suggests that most of the indoor contamination concerns with Mycomycetes that have a particular ability to adapt to the environment inside the buildings. Because fungi concentrations vary over a wide range, threshold values are difficult to determine (Gots et al., 2003). Much data on fungi concentration have been published with respect to home environments. For example, Miller et al. (1988) suggested that fungi concentrations should be below 150 cfu/m3 in home environments. According to Finnish guidelines for urban or suburban residences (Reponen et al., 1992), spore concentrations exceeding 500 cfu/m3 in the indoor air and bacteria concentrations of over 5000 cfu/m3, indicate abnormal microbe sources of indoors.

The present data indicated that culturable air borne fungi were found in almost all homes. Many of the main fungal genera that recovered (Aspergillus, Penicillium, Cladosporium and Alternaria) were similar to those commonly found in residential environments air in other studies conducted in different countries (Li et al., 1995; Garrett et al., 1998; Dharmage et al., 1999; Ren et al., 1999, 2001; Pei – Chih et al., 2000; Duchaine and Meriaux, 2001; Su et al., 2001; Chew et al., 2003; Stark et al.,2003; Horner et al., 2004; O’ Connor et al., 2004 ; de Ana et al., 2006; Lee and Jo, 2006; Claire Dassonville et al., 2008). As previously described (Ren et al., 2001; Chew et al., 2003; Horner et al., 2004; O’Conner et al., 2004; Lee and Jo 2006; Claire Dassonville et al., 2008) species of Aspergillus and Penicillium were found more commonly in indoor air, where as Alternaria and Cladosporium were the next dominant groups other than Aspergillus and Penicillium in outdoors. Other fungal genera such as Curvularia, Fusarium, Mucor, Rhizopus and Trichoderma were less frequently detected in air. In the study of Hyvarinen et al. (2001a), the total concentrations of viable fungi and the concentrations of

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Penicillium and Aspergillus were significantly higher in residences with moisture problems than in the reference buildings. Species of Penicillium have been reported to be associated with moisture damage also in other studies (Li and Kendrick, 1995; Mahooti – Brooks et al., 2004). These findings agree with the results of the present study (i.e.,) increased concentrations or occurrence of the Penicillium spp. were the commonest indicators from indoor air. Higher air borne concentrations of Aspergillus, Penicillium, Cladosporium and non sporing fungi have previously been observed in buildings with moisture damage or visible mould growth (Pasanen, 1992; Pasanen et al., 1992a; Dekoster and Thorne, 1995; Garrett et al., 1998; Salonen, 2007).

The indoor fungal concentration of Aspergillus spp. differs significantly (P<0.05); the mean concentration of 2007 being significantly higher than that of 2008. The means of Aspergillus are significantly, higher than those of other species from the microenvironment of home (Table.16). It also showed that the 2008 mean of Penicillium (9.2 ± 5.5) was significantly (P<0.05) greater than that of 2007 (4.2 ± 5.1) other than Aspergillus. In hotel environment only in the cases of Alternaria, Aspergillus and Penicillium, the respective means of 2007 and 2008 differed significantly. The mean concentration of Aspergillus was significantly higher during 2007 (21.7 ± 11.0) and 2008 (13.8 ± 4.1) (P<0.05) and the mean of Penicillium was significantly higher than other species other than Aspergillus. The present work also reveals, the Aspergillus was the predominant fungus and significantly higher than other species reported from the microenvironments of office premise, public toilet and theatre. However, 2007 mean of Cladosporium was significantly (5.6 ± 3.7) (P<0.05) greater than its 2008 mean (1.5 ± 1.7) in office premise.

Although airborne fungal concentrations were low or high, they were consistent with those reported elsewhere in indoor environments in USA and Canada (Ren et al., 1999, 2001; Duchaine and Meriaux, 2001; Chew et al., 2003; Stark et al., 2003; Horner et al., 2004), in Australia (Garrett et al., 1998; Dharmage et al., 1999; Matheson et al., 2003; Cheong and Neumeister – Kemp, 2005), in Asia (Li et al., 1995; Pei – Chih et al., 2000; Su et al., 2001; Lee and Jo, 2006) and in Argentina (Basilico Mde et al., 2007). However, comparisons must be carried out with care as different sampling methods are used to measure fungal concentrations, air flow rate, duration of sampling, culture agar and in addition, it could also be explained by geographic differences in climate with high humidity (Sometimes up to 90%) and temperature (Dharmage et al., 1999; Matheson et al., 2003). In the present study, the homes were selected randomly and recruited from middle income and low income populations (O’ Connor et al.,

63

2004), so the fungal concentrations were not lower than those described by Clarisse et al. (2007) from a high socio economic status.

The present study revealed the presence of Aspergillus as the predominant fungi in Chennai. Moreover species of Aspergillus, Cladosporium and Penicillium were the most common fungi recovered inside the homes. Tilak and Saibaba (1985) also observed a high concentration of Aspergillus in most indoor environments. They also noted that Alternaria, Cladosporium, Curvularia, Penicillium and Rhizopus were invariably present in all indoor environments. Similar observations were made by Levetin et al. (1978) and Chanbal and Kotmore (1983). Rajan et al. (1952) have recorded Aspergillus as the most common in Kanpur. Thilak and Saibaba (1985) reported Cladosporium to be dominant in Aurangabad. Alternaria was noted to be the predominant type in Jaipur (Gupta et al., 1960) and Poona (Chandbal and Deodikar, 1964). The finding of the present study is in agreement with the findings of several other researchers (Katz et al., 1999; Dharmage et al., 2002; Unlu et al., 2003; Hedayati et al., 2005). Fungi are now seen as having a wider role in respiratory ill health (Li et al., 1995; Dharmage et al., 2002).

These were seven species of Aspergillus with Aspergillus niger predominating. It has been reported that the air inside air conditioned homes has fewer fungi than outdoor air, but has a significantly greater number of species of Aspergillus (Kodama and MacGee, 1986). The poor quality houses had a generally low hygienic standard, a high number of residents, and less provision for effective ventilation systems. The higher number of residents confined to a small space, the higher is the buildup air borne microbes shed by the human body.

The fungal aerosol concentrations in healthy homes were ranging from 10 to 103 cfu/m3 (Solomon, 1976; Hyvarrinen et al., 1993; Kao and Li, 1994; Dekoster and Thorne, 1995) and the literature for mouldy buildings are diverse with some concentrations of 104 cfu/m3 reported (Verhoeff et al., 1992; Hyvarinen et al., 1993). Chew et al. (2003) analysed the level of fungi inside homes, and found that characteristics of the habitat could predict higher or lower concentrations in the dust, depending, for example on the existence of carpets. They also compared the fungal levels in indoor and outdoor air and found Alternaria to be more frequent fungus in outdoor air, while Aspergillus was more prevalent in house.

While evaluating the mould allergy in other indoor environments, Reponen et al. (1992) and Gravesen (1979) indicated that a level of 500 cfu/m3 might be considered as unacceptable and 3000 cfu/m3 of Cladosporium spp. and 100 cfu/m3 of Alternaria spp. may function as a threshold

64 limit for evoking allergic symptoms. It has been reported that a substantial amount of toxins may be absorbed from the inhaled fungal spores (Flanningan et al., 1991). Since recorded toxic fungi from various microenvironments could have contributed toxins, particularly contribution of Aspergillus species, a potent source of the carcinogenic toxin, might be a matter of concern for the workers and the people used to visit places such as hotel, office premise and theatre. The presence of fungi in the atmosphere of hotel is of great concern as the fungal propagules are found to have a great role in food spoilage. They are found to produce off flavours, toxins, discoloration, rotting and are of pathogenic or allergenic nature (Chelkowski, 1991; Bigelis, 1992; Gravesen et al., 1994 and Tipples, 1995). The associated micro flora from different sources, such as citrus fruits, pomaceous and stone fruits, garlic and onion, potato tubers, tomatoes, cereals, nuts, cheese, fats, bread etc., are well defined by Filtenborg et al. (2000). Identification and quantification of large number of species in higher concentrations in hotels is of concern in spoiling the food prepared in those units.

The results of the present investigation have clearly demonstrated that the concentration of air borne culturable moulds, especially Aspergillus and Penicillium is high in the hotels. These are relatively greater risk to work force. The allergenic, toxigenic and pathogenic potentials of these fungi have been well documented in earlier literature (Flannigan et al., 1991). It is necessary to control or minimize the air borne spore levels in hotels. To maintain the hygienic levels in these places: accumulation of moisture in food preparing units should be avoided; indiscriminate throwing of waste in the premises should be avoided; the contaminated food products like vegetables, dough should be destroyed immediately rather than piling them up with in the premises and the floor is to be mopped using antiseptic solution at frequent intervals.

Isolation of large number of species from upholsteries proves that they too serve as a good source for the growth of mould within office premises. It is suspected that moulds from outdoor are found to adhere to the external cover ups of the occupants of buildings and they may get deposited on upholsteries and on carpets which in turn provide favorable niche for their growth. Kemp et al. (2002) reported that furnishings and mattresses without moisture damage can provide a habitat with enough moisture to support fungal growth despite, the lack of an obvious moisture sources.

Menetrez and Foarde (2004) explain the role of heating, ventilation and air conditioning (HVAC) system in the spread of toxic mould spores. The moisture prevents in the filters of AHU and near A/C vent favours the growth of mould spores. The organic stub dropped due to eating

65 and drinking habits of the work force or occupants within buildings provide organic source for their growths in carpets and upholsteries.

An inherent difficulty in conducting airborne microbiotic studies is that there are no recognised health standards such as Permissible Exposure Limits or Threshold Limit Values. At the same time studies have shown that typical air borne microbial levels found in office premises tend to be less than 200 cfu/m3 (Mullins et al., 1976; Sugawara and Yoshizawa, 1984; Hansan, 1986). Because of such findings, it is suggested that levels of microorganisms exceeding 500 cfu/m3 are sufficiently high to warrant an environmental survey; the 500 cfu/m3 value should be incremented to the microorganism’s level in the outdoor site. In such situations, an environmental survey should then be conducted to attempt to locate the predominant sources of amplification of viable particulate for preventive maintenance purposes.

The current fungal aerosol concentrations in homes were ranging from 10 to 104 cfu/m3 which is consistent with other results (Solomon, 1976; Kuo and Li, 1994; DeKoster and Thorne, 1995; Pastuszka et al., 2000). However, the total fungal counts were not as high as the maximum seasonal levels reported in other literature, for example, Solomon (1976) reported maximum of almost 17, 000 cfu/m3 in Upper Silesia, Poland. The current fungal concentrations found in most indoor microenvironments fell within the specified guidelines, between 10 and 1000 cfu/m3, suggested by the American Conference of Government Industrial Hygienists (ACGIH, 1989). However, if the indoor bioaerosol concentration exceeds the guideline then it is recommended that remedial action should be taken to identify the source of emission and methods to reduce the counts. Consequently, the current findings suggest the need for remedial action for indoor microorganisms at the surveyed microenvironment such as home.

Usually fungi produce large amount of spores, which easily become airborne and are able to colonize indoor environments which can utilize nutritional sources and moisture available in indoor materials (Burge, 1992). Indoor spaces with low humidity and characteristic air movements as a result of heating and natural ventilation do not provide favourable conditions for the survival of fungi (Reiss, 1991). In case of sufficient humidity, however fungi may grow on almost all organic substances. Conditions of above 70% relative humidity may be optimal for fungal growth (Burge, 1985).

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5.3. Meteorological studies

For the past 200 years, the field of aerobiology has explored the abundance, diversity, survival and transport of microorganisms in the atmosphere. Microorganisms have been explored as passive and severely stressed riders of atmospheric transport systems. Recently, an interest in the active rolls of these microbes has emerged along with proposals that the atmosphere is a global biome for microbial metabolic activity and perhaps even multiplication. As part a series of papers on the sources, distribution and roles in atmospheric processes of biological particles in the atmosphere, here it is need to be described the pertinence of questions relating to the potential roles that air borne microorganisms might play in meteorological phenomena. For the upcoming era of research on the role of air borne microorganisms in meteorological phenomena, one important change is to go beyond descriptions of abundance of microorganisms in the atmosphere towards an understanding of their dynamics in terms of both biological and physico - chemical properties and the relevant transport processes at different scales. Another challenge is to develop this understanding under contexts pertinent to their potential role in processes related to atmospheric chemistry, the formation of clouds, precipitation and radiative forcing.

In the present investigation, all the microbial concentrations measured in both the outdoor and indoor air depended strongly on the season. Summer was generally found to have higher indoor microbial concentration than winter. This seasonal trend for the bacteria and fungi was consistent with previous indoor measurements (Pastuszka et al., 2000), except there was no seasonal difference in the bacterial concentrations in the previous indoor measurements. The temperature and relative humidity measured along with the microbial measurements in the current study revealed that the summer values for both the outdoor and indoor air were relatively higher (Table 2&14), meantime during the month of January (winter) both bacterial and fungal counts of both the environments were also found to be higher. Typically a higher environmental temperature and relative humidity favour microbial growth (Ren et al., 2001). Accordingly, it is suggested that the temperature and relative humidity were important factors causing the seasonal difference in the microbial concentrations.

Supporting to the statement, a higher total bacterial count (cfu/m3) was observed during the month of January (winter) and a lower count was noticed during the month of October, 2007 and December, 2008 from outdoor microenvironments (Table 2). In contrast to this, indoor total bacterial counts were found to be higher during April, 2007 and March, 2008 and a lower count

67 was observed in July, 2007 and December, 2008 (Table 10). Staphylococci, Micrococci and Bacilli were the dominant groups. Staphylococci were found to be present throughout the study period. This may be due to the favorable mean monthly temperature 30.4° C (2007) and 29.7° C (2008) during the month of January (Table 17) which supports the growth of bacteria. In addition to this, relative humidity 77% (2007) and 80% (2008) is an important factor to increase the bacterial load. The mean monthly wind speed also showed little influence in bacterial concentrations.

The occurrence of Aspergillus was a major factor in the seasonal variation in the number of fungal propagules in outdoor and indoor air. In the current study, Aspergillus was observed throughout the year with mean monthly concentration 18.3 ± 8.4 (2007) and 17.5 ± 5.1 (2008) reckoned in the month of June (summer) and February (spring) respectively in current study (Table .19a). The distribution of Aspergillus was also higher during winter (December and January) which was supported by Fernandez et al. (1998) in Leon, noted that the spores of Aspergillus were prevalent in Winter. This was also followed by Penicillium, the second predominant fungus.

Rainfall and relative humidity almost always have profound effects on the level of fungi. It has been stated that Alternaria level may decrease in winter as opposed to Penicillium and Aspergillus levels which may be high in spring and fall, despite the fact that they may be found in the atmosphere all year round (Al – suwaine et al., 1999). The present study revealed that the total number of fungal colonies increase in winter which is consistent with the results of other studies. ( Al – suwaine et al., 1999). Although some differences were observed between the two years with respect of the cfu/m3 and average monthly concentrations, but no statistically significant difference was observed when the total over all yearly concentrations were considered. The possible reason might be the insignificant changes of environmental factors during the two sampling years.

5.4. Prevalence of Aspergillus in various microenvironments: Moulds produce acute health effects through allergy or infection. Hypersensitivity pneumonitis, a particular form of granulomatous lung disease, is a syndrome caused by inhalation of large concentrations of dust containing organic material including fungal spores. It is generally an occupational hazard in agriculture, but has been reported in individuals exposed in home (Flannigan et al., 1991). Other symptoms include head ache, dizziness, dermatitis

68 diarrhoea and impaired or altered immune function. Indoor fungal allergens probably affect a significant proportion (10 – 32%) of all asthmatics are sensitive to fungi. Opportunistic fungal pathogens such as Aspergillus are common in indoor air. A normal, healthy individual can probably resist infection by these organisms regardless of dose, although high exposures may cause hypersensitivity pneumonitis. However, any mould that can grow at body temperature can become a pathogen in an immuno – compromised host. Individuals undergoing chemotherapy, organ or bone marrow transplantation or those with HIV/AIDS are especially susceptible to invasive infection by species of Aspergillus (Sandra et al., 2003).

The present results showed a significant increase in fungi isolated from the indoor air. The identified genera suggest a mix contamination and the dominant colonies isolated from the air (Home) were Aspergillus, Penicillium, Cladosporium, Alternaria, Rhizopus and Mucor. Aspergillus and Penicillium were isolated more frequently than other fungi. Aspergillus was the most common fungus in indoor and outdoor probably due to their thermo tolerant ability. The most common air borne fungi encountered indoor nearly paralleled to those found outdoors. Concerning percentages of Aspergillus isolated from various microenvironments, the vegetable market recorded the highest percentage (41.6) among the outdoor microenvironments whereas, a very high percentage distribution (48.6%) of Aspergillus in home and a less distribution (25.1%) in office premise were observed in indoor microenvironments. The total count of Aspergillus from indoor environments showed 36.8% where as outdoor seems to be 33% (Table 18) in two year study period.

Exposure to some fungi can induce allergic or asthmatic reactions, while other species can cause primary infectious diseases. Affected individuals often experience relief when they leave the building for several days (Bush, 1989). Miller et al. (2000) studied the extent and nature of fungal colonization of building materials in 58 naturally ventilated apartments that had suffered various kinds of water damage in relation to air sampling done before the physical inspections. Approximately 90% of the apartments that had significant amounts of fungi in wall cavities were identified by air sampling.

Ren et al. (1999) characterized the nature and seasonal variation of fungi inside and outside homes in the Greater New Havan. No significant difference in concentration and type of fungi between living room and bed room or by season was observed. Penicillium and Aspergillus were dominant in indoor air during January (winter) but were equally dominating in almost all seasons from outdoor air. Air sampling in every suspected house is suggested for year

69 round fungal exposure assessment. The present result also showed the highest monthly mean concentration (cfu/m3) of 18.3±8.4 during the month of June (summer) for the year 2007 and lowest of 5.8 ± 5.0 (cfu/m3) during the month of September (fall) for the same year (Fig. 21). This is supported by many other workers and concluded that due to their thermo tolerant ability, Aspergillus is the most common fungus in indoor. The distribution of Aspergillus was slightly lower during 2008 than 2007. However, the highest monthly mean concentration of 17.5 ± 5.1 (cfu/m3) was observed during the month of February (spring) and less concentration of 11.5±5.0 (cfu/m3) was noticed during May 2008 (summer) (Table19.a).

Pei – Chih et al. (2001) evaluated the air borne fungal concentrations at urban and suburban areas in Taiwan. In summer, the total fungal concentration, both indoors and outdoors of suburban homes, were significantly higher than those of urban homes. Shelton et al. (2002) examined 12, 026 fungal air samples from 1717 buildings in United States and found the most common culturable air borne fungi, both indoors and outdoors and in all season were Penicillium, Cladosporium, Aspergillus and non sporulating fungi. This result also supports the present work where the same type of culturable air borne fungi was obtained. The statistical analysis for the annual concentrations (cfu/m3; Mean ±SD) of Aspergillus from various microenvironments of outdoor and indoor was carried out for the year 2007 and 2008 and compared under parametric and non parametric test (Fig.19b). From the atmosphere of home, the annual concentrations of 26.7 ± 7.5 (2007) and 19.8 ± 9.2 (2008) were noticed as the highest and found to be significant (P<0.05) both in t – test and Mann - Whitney test. In general, Aspergillus was the only predominant fungus present almost in all the microenvironments. (Table 19b).

More than 80 genera of fungi have been associated with symptoms of respiratory tract allergies (Horner et al., 1995). Aspergillus, Alternaria, Cladosporium and Fusarium are amongst the most common allergenic genera. Metabolites of fungi are also believed to irritate the respiratory system. Furthermore non biological particles may serve as carriers of fungal allergens molecule into the lung independently of the whole fungal spores. Allergenic molecules could conceivably be carried into the lung at a greater depth than a fungal spore would be expected to penetrate (Lippman et al., 2003). Airborne bacteria and fungi can be the cause of a variety of infectious diseases as well as allergic and toxic effects. Particles smaller than 5 μm, the so called respirable fraction, are able to penetrate into the alveoli and can lead to allergic alveolitis and other serious illnesses (Lacey and Croock, 1988; Chatigny and Macher, 1989; Burge, 1990; Owen et al., 1992; Seltzer, 1995)

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Inhalation of mould spores and hyphal fragments commonly leads to allergy, especially to asthma (Gravesen, 1979; Braback and Kalvesten, 1991; Ninan and Russel, 1992; Dutkiewicz, 1997). Among the respiratory symptoms reported has been nasal congestion or runny nose, shortness of breath and wheezing (Goldfarb, 1968; Platt et al., 1989; Strachan and Sanders, 1989). Also a study carried out in Sosnowice, located in Upper Silesia, Poland showed that asthmatic children very often live in dwellings belonging to a group of homes with an elevated level of airborne respirable particles and fungi (Pastuszka et al., 1998). Therefore, in mouldy homes the risk of asthma for its residents, associated with an exposure to the total mould or to some specific genera, probably increases with the inhalation of fungal particles as well as their products (voltaic organic compounds, toxins, glucans). However, the lacks of relation between the occurrence of the respiratory symptoms, including asthma and the measured concentrations of bioaerosols have been found by others. Dotterud et al. (1996) observed that prior to asthmatic attacks the child could always notice a smell of mould but when sampling the air only a few spores were found. Some fungi, for example Mucor are more often found in floor dust and carpets than in air samples (Gravesen et al., 1994; Koshinen et al., 1995 and Gravesen, 1978, 1979) studied respiratory symptoms and infections among children in a day care centre with mould problems. Mouldy growth on the walls also adds to the number of allergenic spores in the air within the building. It has been stated that an average concentration below 500 allergenic spores/m3 air causes only minor symptoms in persons known to react very strongly to allergenic species while concentrations above 500 or 600spores/m3 induce symptoms of disease in all person who suffer from allergy (Rapiejko, 1997).

5.4.1. Immunological studies

Mainly, but not exclusively, exposure to pollutants from biological origin (Edwards, 1980; Patterson et al., 1981) promote illnesses and irritant, toxic and allergenic symptoms (Flannigan et al., 1990; Sorenson, 1990; Strachan et al., 1990; Su et al., 1992). The majority of the health effects linked to dampness and moisture of buildings are those of the respiratory system. Excess humidity promotes the growth of microorganisms such as moulds and bacteria that lead to release of pollutants into indoor air. Allergy is one of the health problems in the world and serum IgE level is a means of diagnosis of allergy (Host and Halken, 2005; Dennis, 2003). Chan and Mckenzie (2003) reported that total IgE levels in healthy subjects are higher than the previous references argued that the pollution may be the cause of this elevation. It was observed that IgE levels in Indian population were relatively higher than the western values (Chowdary et al., 2003). According to Wittig et al. (1980) healthy, non allergic adults have an

71 expected IgE concentration up to 120 IU/ml. The higher IgE levels in normal controls in India are explained probably by the higher incidence of parasitic infestations.

Atopy is a tendency to produce excessive amounts of IgE antibodies when exposed to allergens (Burrows et al., 1989). IgE is produced by B lymphocytes moreover a trace protein and normally accounts for less than 0.001% of total serum immunoglobulin. The concentration of IgE in serum is age dependent and normally remains at levels less than 10 IU/ml in most infants during the first year of life. There is a wide distribution of expected serum IgE values in healthy individuals of same age group (Kjellman et al., 1976). An atopic individual responds to antigenic stimuli to which normal people will not respond. B lymphocytes and plasma cells in airways, gastro intestinal tract and regional lymph nodes produce IgE. The initial formation of IgE antibody depends upon the signals from lymphocytes and IL – 4 and IL – 13. The molecular mechanism underlying (responds and the subsequent production of IgE antibody) immune system activation for allergen induced asthma includes stimulation of CD4+ Th2 immune response and the subsequent production of IgE antibody. Re - exposure to allergen results in the recruitment of mast cells (via high affinity IgE Fc receptors), eosinophils and other leukocytes. In particular, mast cells that release the vaso active amines, histamine and other ligands from large granules produce a local systemic hypersensitivity reaction (Janeway et al., 2001). The ensuring inflammation amplifies an individual’s hypersensitivity reaction by the recruitment of other cells and perpetuates the clinical symptoms (wheezing, shortness of breath, and chest congestion) (King, 1999). In atopic individuals, the IgE receptors send unusually strong signals when cross linked, resulting in secretion of abnormally high levels of IL – 4 from mast cells, which further results in over production of IgE antibodies.

According to Halonen et al. (1982), a significant relationship exist between serum IgE levels and eosinophilia in individuals where IgE levels presumably provide a better clue to allergy than do skin test. Yamada et al. (1998) were of the opinion that detection of specific IgE is a prerequisite for both, the definitive diagnosis and the therapeutic strategy of allergic disorders. Di Lorenzo et al. (1997) reported that there is an interrelationship of the allergen type, total serum IgE, eosinophil and bronchial hyper responsiveness suggesting that all three may play role in development of allergic disorders. Fahy (2000) stated that IgE secretion by lymphocytes defines the allergic state and nearly all asthmatics have a higher IgE levels in serum than normal’s, following adjustment with age and sex. Depending on their age children may be more vulnerable than adults to air pollutants. The general presumption that fungi induced allergy is associated with peripheral eosinophil which did not correlate with our findings. In fact, about

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106 out of 115 subjects showed peripheral blood eosinophil counts within the normal range. Though, differential eosinophil count was used for evidence of allergy, in our study, it did not give satisfactory information about allergy state because it was negative in more than 90% of the subjects studied.

Interpretation of IgE antibodies as biomarkers of exposure is problematic, because such antibodies last in the blood for months, thus it is not known to what extent IgE levels reflect the magnitude of exposure. The use of raw OD values only allows comparisons to be made within a single assay and does not address the problem of sera assayed at different dilutions. Misra et al. (1983) used a system of a cut off OD values related to negative controls and factor to define positivity. Increased IgE concentration in allergic aspergillosis has been reported by Heiner and Rose (1970) as being in excess of 800 ng/ml and by Patterson et al. (1973).

The analysis of results can vary considerably depending on whether the whole taxa (genera) are considered or the analysis carried out according to species (Horner et al., 2004). Specific antigens, that are usually the most important allergens, are not always shared by the species of the same genera. Hence, in environmental mycological studies, it is advisable to identify the species in order to obtain valid data that correlate with levels of sensitization and clinical manifestations. The potential role of specific IgE antibody against Aspergillus has still to be investigated. This points out the interest of quantitative measurements of IgE antibody. Various studies show that IgE antibody is directed against the antigenic fraction of the fungus that supports cross reactivity with other Aspergillus species, a fact which may explain the common features of immediate type hypersensitivity associated with infection by the various Aspergillus species (Dessaint et al., 1975). There are no international standardized species – specific control sera available. Recent studies showed that the level of IgE in normal individuals was significantly higher than that was reported previously (Chan and McKenzie, 2003). There are some possible factors, such as parasitic diseases, smoking, alcohol drinks and malnutrition that may increase total IgE serum level (Finkelman and Urban, 2001; Forte et al., 2003). Therefore, defining a reference range in normal subjects for IgE level is very difficult (Klink et al., 1990). In the present study normal values of total IgE in males were relatively higher than females, similar to the study reported by Berciano et al. (1982). The total IgE levels were elevated in 18.3% (21 of 115) subjects with allergic disorders. The value was about 1000 IU/ml at the maximum (Table.24), when this was associated with fungal elements (Aspergillus induced allergy), the IgE values ranged from 500 IU/ml to more than 2000 IU/ml (Fahy, 2000) (Fig.25). The total IgE levels obtained here as reference ranges in individuals from a microenvironment of

73 home might be useful for the diagnosis of allergic disease. However, due to the variation of allergens in cities and air pollutions, it is recommended to monitor local normal range of serum total IgE every ten years. Based on the present study, it is recommended therefore that total serum IgE levels and peripheral eosinophil counts to be done in all subjects along with other investigations like specific serum IgE level in Indian population.

More females than males develop asthma during puberty, so prevalence of adult asthma becomes higher in females than males (Bapna et al., 1998). More than 50% of the subjects in this study had any one of the allergic symptoms before the age of 40 years. This is in accordance with other studies, which have been shown that, in the majority of subjects, with extrinsic allergy, the symptoms develop before the age of 30 years (Duane et al., 1995).

When comparing the severity of allergy with serum IgE levels in subjects from a home environment, the present data indicated that the more severity of allergic disorder, the greater is the elevation in serum IgE. The most important risk factor for the development of extrinsic allergy is atopy (Lebowitz et al., 1984; Peat et al., 1996). The basic pathology is hyper responsiveness. The airway response is an excessive response of the airway epithelium to antigenic stimuli. This response is mediated by T – Lymphocytes. Antigenic exposure to T – lymphocytes leads to their differentiation in to active T – cells, which secrete a series of biologically active proteins called cytokines. The secretion of IgE by lymphocytes defines the allergic state of an individual. The cellular events associated with IgE dependent processes are very much important in asthma (Villar et al., 1985). Higher IgE levels indicate some types of inheritant susceptibility and/or presence of a disease process involving airway inflammation (Sherril et al., 1995; Chowdary et al., 2003; Anupama et al., 2005).

The clinical syndrome produced by an allergic reaction depends critically on three variables; the amount of allergen specific, the root by which the allergen is introduced and the dose of allergen.

Anaphylaxis is an acute, systemic, hypersensitivity response to allergen which typically involves multiple organ system and which if untreated can lead rapidly to death. Allergic rhinitis results if allergen is introduced through airway through inhalation symptoms which includes sneezing, nasal congestion and itching, rhinorrhea, probably primarily reflect IgE dependent release of mediators by effector cells in response to aeroallergens (Richard deshazo., 2000).

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Asthma affects millions of people worldwide and allergic asthma is triggered by allergen induced activation of sub mucosal mast cell in the lower airways. Allergic disorder that are associated with IgE not only causes significant morbidity and lost productivity, but also are increasing in incidence and in human and economic impact in many parts of the developed world. In recent years, the increasing incidence of allergy is well recognized not only in the developed but also in developing countries across the globe. Allergy can develop at any age and heredity plays a key role in who will develop it. The likelihood that a given individual will develop an allergic disease reflects a combination of genetic and environmental factors.

5.5. Air sanitation

No other area in occupational and environmental health has experienced such rapid changes in the recent past as has indoor air quality. Due to an emphasis on energy conversation buildings are more dependent on mechanical ventilations systems for the delivery of fresh air. At the same time, the use of building furnishings maid from synthetic chemicals has increased, as has the recognition of microbiological organisms as potential exposure hazards. The sum result of these factors has been a steady increase in the frequency of indoor air quality complaints. No standard method has been adopted for evaluating air sanitizers.

Air fresheners are rarely necessary because they cannot substitute for good ventilation; the best solution is to open windows to bring in fresh air or to use fans to maintain air circulation. Air fresheners also are not a solution to poor quality, they mask bad odours but they do not eliminate the chemicals that cause them.

Of all the products in home, clean smelling air fresheners seen to pose little risk. But the fresh scent of air fresheners may mask a health threat – chemicals called phthalates that can cause hormonal abnormalities, birth defects and reproductive problems. To protect consumers, government regulators should follow up by doing more thorough tests on these products and enacting basic measures to limit exposure to these chemicals. Meanwhile, consumers may wish to avoid using air fresheners especially in places where there are children or pregnant women. Hence, the Consumer Product Safety Commission should ban hazardous phthalates in consumer products and should require that manufacturer provide ingredient information on the label. In particular, the European study detected cancer causing chemicals such as benzene and formaldehyde in some air fresheners. Benzene is known to cause leukaemia in humans and formaldehyde has been linked to cancers of the upper airways. People with allergies to these

75 chemicals could have adverse reactions, including rashes or even asthma attacks from exposures to products (SCHER, 2006).

There is considerable evidence that glycol vapour produce significant decreases in numbers of viable airborne bacteria to maintain suitable concentrations in the air of enclosed spaces. Several investigators have shown that glycols (triethylene, dipropylene or propylene glycol) at concentrations of 5% or more in such formulations will temporarily reduce numbers of airborne bacteria when adequate amounts are dispensed under relatively ideal conditions.

In order to reduce bioaerosol loads in indoor environments, certain control measures can be followed. These include proper identification and elimination of the microbial source in occupational and house hold settings, maintenance of equipments, humidity control, natural ventilation, use of filters in ventilation and air cleaning by the use of disinfectants, air sanitizers and biocides. Periodical use of disinfectants and biocides is one of the methods to ensure controlled bioaerosol concentrations.

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6. SUMMARY

It is important to note that geography and climate play an important role in determining the microbial concentrations because the transport of bioaerosol is primarily governed by hydro dynamic and kinetic factors, while their fate is dependent on their specific chemical makeup and the meteorological factors to which they are exposed. The presence of people is the most significant parameter resulting in elevated indoor bioaerosol counts in the absence of significant indoor or outdoor sources. Hence, it is essential to evaluate the quality of air we breathe, whether indoor or outdoor. The number of airborne microorganisms can indicate the degree of cleanliness and may be a source of human discomfort. The results of the present work have clearly demonstrated various outdoor and indoor microorganisms at different microenvironments and found that bioaerosol counts are an integral part of the indoor pollution characterization puzzle and important for quantification of airborne allergens and pathogens.

The present study measured the total bacterial and fungal concentrations in the outdoor and indoor air of various microenvironments under uncontrolled environmental conditions from January 2007 to December 2008. A total of 10 microenvironments (Outdoor – bus stand, railway station, recreation ground, sewage treatment plant and vegetable market; Indoor – home, hotel, office premise, public toilet and theatre) were selected for the study. In a microenvironment, 12 samples were surveyed at regular intervals of a month in a year. A total of 5495 cfu/m3 of bacteria was obtained in outdoor environment, of which 3032.5 (55.2%) and 2462.5 (44.8%) were observed during the year 2007 and 2008 respectively. Air samples in an outdoor microenvironment of recreation ground were observed a highest number of total bacteria (1332.5; 24.2%) in the two year study period.

A total of 4845 cfu/m3 of fungi was obtained in outdoor air, of which 2347.5 (48.4%) and 2496.5 (51.5%) were observed during the year 2007 and 2008 respectively. During the study period, air samples in a microenvironment of bus stand were observed a highest number of fungi (1045; 21.6%). In general, the outdoor air concentrations of total fungi were lower than the concentrations of the total bacteria. Of 4902.5 (cfu/m3) total bacteria in indoor air 2787.5 (56.8%) and 2115 (43.1%) were obtained in the year 2007 and 2008 respectively. In the microenvironment of home, a highest number of bacteria (1200; 24.5%) was obtained throughout the study period.

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About 5055 cfu/m3 of fungi was obtained from the indoor environment, of which 2837.5 (56.15%) and 2217.5 (43.9%) were obtained during the year 2007 and 2008 respectively. A total of 1145 cfu/m3 (22.7%) was the highest in home among the indoor microenvironments. In general, indoor fungal concentrations were higher than the concentration of the indoor bacteria.

The month wise distribution of bacterial and fungal concentrations was noted to vary during each month in all the sampling environments and was not uniform throughout the study period. There was a less difference between outdoor and indoor bacterial counts in month wise distribution, ranging from 10.4% to 6.5% (outdoor) and 10.0% to 7.1% (indoor).

In extramural air, 8 different bacterial genera were studied, of which 3 of Gram positive (3125; 56.9%) and 5 were Gram negative (1510; 27.5%) types. Species of Staphylococcus and Bacillus were found to be the most prevalent bacteria and others such as species of Micrococcus, Aeromonas, Escherichia, Pseudomonas, Klebsiella and Serratia were also identified. In general, the concentrations of species of Staphylococcus and Micrococcus were significantly (P<0.05) greater in outdoor microenvironments. Staphylococcus was the predominant bacterial type in almost all outdoor air studies and had the highest count, constituting 21.6% of the total bacterial genera whereas the second predominant group was Bacillus, constituting 18.5% and the third Micrococcus of 17.2% of total.

A total of 9 different bacterial genera were identified in intramural air, of which 3 and 6 were Gram positive (3060; 62.4%) and Gram negative (1147.5; 24.2%) bacterial types respectively. Species of Staphylococcus and Micrococcus were the predominant bacterial groups. Species of Bacillus, Aeromonas, Escherichia, Pseudomonas, Klebsiella, Serratia and Proteus were also identified among the genera.

Species of Staphylococcus were present in almost all the indoor microenvironment studies; these bacteria had the highest count, constituting 26.6% of the total bacterial genera followed by Micrococcus constituting 24.1% of the total indoor concentration. These two bacterial genera were found to be significant (P<0.05) in indoor air.

Of the 9 different fungal genera studied, species of Aspergillus (1600; 33.0%) were found to be the most prevalent organisms in the outdoor air. Species of Penicillium and Alternaria were also found to be the second and third respectively. Organisms such as species of

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Cladosporium, Curvularia, Fusarium, Mucor, Rhizopus and Trichoderma were also identified among the examined groups.

The outdoor fungal concentrations (Mean ± SD) of Aspergillus in 2007 and 2008 was significantly (P<0.05) greater than those of other organisms in the corresponding years from the microenvironment of bus stand, railway station, recreation ground, sewage treatment plant and vegetable market. Aspergillus seems to be the predominant fungus followed by Penicillium in 2007 and 2008 which were significantly (P<0.05) greater than those of the other species in the microenvironment of bus stand, sewage treatment plant and vegetable market.

The type and total number of fungal isolates were identified and grouped into 9 different genera from the intramural environment. A total number of 1865.5 cfu/m3 (36.8%) of Aspergillus spp. and 647.5 cfu/m3 (12.8%) of Penicillium spp. were found to be the most prevalent groups. The indoor fungal concentration of Aspergillus spp. differs significantly (P<0.05), the mean concentration of 2007 being significantly higher than that of 2008. Like outdoor environments, indoor concentration (Mean ± SD) of Aspergillus significantly greater than (P<0.05) than those of other species from the microenvironments of home, hotel, office premise, public toilet and theatre.

Correlation between monthly bacterial and fungal loads with various meteorological factors such as temperature (ºC), relative humidity (%), total rainfall (mm) and wind speed (km/h) were studied. No statistically significant difference was observed in the total overall yearly concentrations. The possible reason might be the insignificant changes of environmental factors during the sampling years.

The distribution percentage and total number of Aspergillus in various microenvironments were analyzed and a total of 1600 cfu/m3 in outdoor and 1862.5 cfu/m3 in indoor air were obtained. Among the outdoor microenvironments, vegetable market showed the highest percentage of 25.32 cfu/m3 and home environment had the highest percentage of 29.93 cfu/m3 of Aspergillus among the indoor microenvironments. There was no much difference in percentage distribution of Aspergillus in other microenvironments.

The highest monthly mean Aspergillus concentration (cfu/m3) of 18.3 ± 8.4 was observed during the month of June (summer) in 2007 and 17.5 ± 5.1 (cfu/m3) in February (spring), 2008. The annual concentrations of 26.7 ± 7.5 (2007) and 19.8 ± 9.2 (2008) were noticed the highest

79 and found to be significant (P<0.05) in home environment. In general, Aspergillus was the only predominant fungus present in almost all the microenvironments.

The relation between the microbial air quality and allergic status of the selected population in the sampling site (home) were evaluated. A total of 115 individuals (46 males and 69 females) from the volunteers in home environment were recruited as case study for the total immunoglobulin E level. 25 healthy individuals (11 males, 14 females) were included as control study.

Majority (32.2%) of case study subjects were studied from the age groups ranging between 21 and 30, and between 31 and 40 years, whereas a highest total of male (15 of 115; 13%) subjects were found to be the age group ranging between 21 and 30 years and female subjects (24 of 115; 20.9%) were observed in the age group between 31 and 40 years.

The percentage distribution of eosinophil was analyzed for the preliminary identification of allergic status. About 92.2% showed peripheral blood eosinophil counts within the normal range whereas 6.1% and 1.7% showed an elevated and a high eosinophil range respectively. The general presumption that fungi induced allergy is associated with peripheral blood eosinophil did not correlate with the present findings.

The most common invitro test for assay of allergen activity is Enzyme Linked Immuno Sorbent Assay (ELISA) and was performed to study the immunoglobulin E antibody level as the serological index to relate with the allergic status. About 18.3% of the subjects had an elevated level of total IgE (> 160 IU/ml) whereas about 81.7% of the subjects showed no significant difference between the means of the different groups (< 160 IU/ml). The total IgE levels obtained here as reference ranges in individuals from a microenvironment of home might be useful for the diagnosis of fungal allergic disease.

The potential role of specific IgE antibody against Aspergillus was estimated by ELISA quantification method to study the fungal allergic status in study population. About 2.6% of the subjects had specific IgE level and the highest level of specific IgE was found to be ranging between 641 and 800 IU/ml. There are no internationally standardized species specific control sera available.

A mouldy home, the risk of asthma for its residents, associated with an exposure to the total mould or to some specific genera, probably increases with the inhalation of fungal particles

80 as well as their products. To remediate the high risk site (home), a suitable method was employed by appropriate air sanitation method to check its efficacy. Bacillocid is the commonly used, commercially available surface and environmental disinfectant that has very good cleansing property along with bactericidal, fungicidal, viricidal and sporicidal activity. The application of Bacillocid does not require shut down of the contaminated areas for 24 h with a viable count reduction of 99.9% over the parallel untreated control, after correcting for settling rates, in the air of the test enclosure.

The likelihood that a given individual will develop an allergic disease reflects a combination of genetic and environmental factors. But then, the knowledge on the presence of mould within indoor air is important to take remedial measures and provide relief to the people involved in the environments.

The present study suggests that the city of Chennai harbours various species of bacteria and fungi due to its warm and slight rainy climate and very rich flora. It is of significance that our findings may be of use with regard to the diagnosis and prophylaxis of allergic diseases thought to be resulting from air borne fungi, when using allergic tests the spectrum of the fungal genera examined in the selected environments (Home). This study may thus be of considerable assistance to scientist and clinicians working in this field in adopting preventive measures and/or selecting an appropriate antigen for diagnostic purposes.

The complexity of carrying out similar studies highlights the need for establishing valid approaches that would contribute and add to our aerobiological, epidemiological and clinical knowledge. The following recommendations are the outcome of the present study. x A routine programme of building inspection and assessment can identify problems before complaints and building related symptoms occur. x Monitoring of indicators other than concentrations may be helpful, for instance ventilation rates, general cleanliness and signs of dampness. In addition, it is necessary to investigate how people are exposed to pollutants in indoor air and how the exposure levels could be measured or estimated using computer models. x Persons residing in air conditioned homes may have a higher frequency of respiratory complaints than those living in naturally ventilated homes.

81 x The air conditioning ducts should be kept free from dirt and spores. Any leaks in air conditioning ducts which might introduce dirt and should be checked and repaired. x Manual scrubbing with brushes and flexible rods and whips and can assist in cleaning hard to reach dirt build up in air handling systems. x Some commercial air cleaners are equipped with tiny fibre – optic cameras to assist in locating dirt build up and to confirm cleaning effectiveness. x The exhaust ventilation fans could reduce the microbial counts and other air borne contaminants including volatile organic compounds and gasoline vapour, which directly or indirectly affects health. This intervention is simple and easy to implement because it does not require more resources or skills. x Concentrations can be achieved by installing this new generation of hybrid air filters. Engineering control methods must be balanced with constraints such as occupant comfort, economic factors and building management strategies to ensure that the health risks associated with bioaerosol exposure are as low as practical. x The use of HEPA filtration can be used to trap very small particles. x Chemicals should also be applied to indoor air. The use of sanitizing solutions to kill mould and bacteriological growth opens the question of safety for humans and pets in the air conditioned area. x Visible mould can be removed by disinfection with a chlorine bleach solution. The area being cleaned should be well ventilated, as chlorine itself is volatile and irritating. x Proper identification and elimination of the microbial sources in occupational and house hold settings, maintenance of equipment, humidity control, natural ventilation, use of filters in ventilation and air cleaning by the use of disinfectants and biocides. x More research and data are needed, particularly on particles and microbes, volatile organic compounds from consumer products, building dampness, levels of exposure and effects on vulnerable populations. x All possible routes of exposure should be considered. Health based guideline values for key pollutants and other practical guidance should be developed.

82 x The impact of indoor exposure should be considered when evaluating the health effects of outdoor air pollutions. All relevant sources known to contribute indoor air pollution should be evaluated. A reasonable reduction in the bioaerosol adds to our aerobiological, epidemiological and clinical knowledge.

83

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APPENDIX - I

Composition of Bacillocid (Each 100gram contains) g

1, 6, Dihydroxy 2, 5 – Dioxy hexane 11.2

(Chemically bound formaldehyde)

Glutaraldehyde 5.0

Benzal konium chloride 5.0

Alkyl urea derivative 3.0

Preparation (manufactured by Raman and Weil private limited, Bombay, India)

BacillocidR spl, 50ml was mixed with 10 litres of water to get 0.5% solution. Composition of Nutrient agar g/L

Peptic digest of animal tissue 5.0

Beef extract 1.5

Sodium chloride 5.0

Agar 15.0

pH 7.4 ± 0.2

Composition of Blood agar g/L

Infusion from beef extracts 50.0

Tryptose 1.0

Sodium chloride 0.5

Agar 15.0

pH 7.3

The above ingredients were dissolved, autoclaved and cooled to 45 to 50°C. To this 5ml of sterile defribrinated sheep blood was added aseptically. This was mixed thoroughly to avoid accumulation of air bubbles and dispensed into sterile petriplates.

Composition of MacConkey agar g/L

Peptic digest of animal tissue 20.0

Lactose 10.0

Bile salts 5.0

Sodium chloride 5.0

Neutral red 0.07

Agar 1.5

pH 7.5 ± 0.2

Composition of Potato dextrose agar g/L

Potato infusion 200.0

Dextrose 20.0

Agar 15.0

pH 5.6 ± 0.2

Composition of Lactophenol Cotton blue g/L

Phenol 10.0

Lactic acid 10.0

Glycerol 20.0

Cotton blue 1.0

Composition of Leishman’s stain g/L

Leishman’s stain powder 1.5

Methanol 1000.0