ARBOVIRAL outbreak


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ARBOVIRAL OUTBREAK A DEADLY SCOURGE Role of the Microbiologists in the investigation of an outbreak:

ARBOVIRAL OUTBREAK A DEADLY SCOURGE Role of the Microbiologists in the investigation of an outbreak Dr K.Prasanthi , MD Department of Microbiology, Gandhi Medical College , Secunderabad INDIA Dr.K.Prasanthi MD 1


Introduction Arboviruses contributing to a large proportion of total burden of infections Viruses are entering into new territories , are more virulent than earlier and becoming endemic in new regions Ex :Chikungunya ,Dengue , West nile, JE , KFD, Chandipura which were almost not existing in India , but now reported from all parts of the country Severity of these illnesses is totally depends on the efficacy of the surveillance system Dr.K.Prasanthi MD 2

Perspectives :

Perspectives Arboviruses : large group (more than 400) of enveloped RNA viruses which are transmitted primarily (but not exclusively) by Arthropod vectors (mosquitoes, sand-flies, fleas, ticks, lice, etc) Humans are infected only if they get in the way of the viral natural cycle by entering areas where the viruses are prevalent and being bitten by an infected arthropod Infection is often in apparent or trivial but some of these viruses can cause very severe , even fatal illnesses including febrile illnesses often with rashes and arthritis, infections of the nervous system and hemorrhagic fevers Dr.K.Prasanthi MD 3

What is an Outbreak:

What is an Outbreak Appearance of an unusual number of cases of a disease or condition in a population in a given period of time and place Outbreaks of Japanese Encephalitis were reported largely from India and Nepal There were also several outbreaks of Dengue, Chikungunya and JE in recent years Resulted in considerable morbidity and mortality Dr.K.Prasanthi MD 4


INDIAN SCENARIO An epidemic of viral encephalitis was reported in 2005 in India. Where 5,737 persons were affected and 1,344 persons died. More than 60 outbreaks of Dengue have occurred since 1956 There was a huge outbreak of Dengue in 2003 with 12,754 cases and 215 deaths In 2006 outbreak it was estimated that 10,344 cases and 162 deaths due to severe forms of Dengue (DSS/DHF) had taken place. Chikungunya fever , which is responsible for significant human morbidity for several years, was first reported in 1963 in Calcutta followed by epidemics in Tamilnadu, Andhra Pradesh ,ending in Maharashtra in 1973 after 8 years of quiescence After 32 years in 2005 the disease has reemerged causing 1.3 million cases in 13 states of India including Andhra Pradesh Dr.K.Prasanthi MD 5

Preparedness: An Essential Element:

Preparedness: An Essential Element Because widespread epizootic activity , larger outbreaks of arboviral infections and human illness are possible if adequate surveillance, prevention activities and mosquito control measures are not established and maintained Private practitioners , the major service providers in India and a frontline to control many diseases are failing to guide people to take preventive measures Dr.K.Prasanthi MD 6

Preparedness Is Essential:

Preparedness Is Essential Integrated and accelerated action to reduce mortality by these threatening diseases is done by strong interaction, commitment , knowledge, training of various health departments in the investigation of any disease outbreak The involvement and expertise of infectious disease physicians, microbiologists, and public health practitioners are essential to the early detection and management of epidemics Dr.K.Prasanthi MD 7

Slide 8:

Although clinical diagnosis is useful, Laboratory confirmation will be more meaningful . Rapid alert to outbreak situations may be accelerated with microbiology laboratories integrated into national and supra-national global surveillance programmes Dr.K.Prasanthi MD 8

Causes of Arboviral epidemics:

Causes of Arboviral epidemics Travel of susceptible individuals into an endemic area where the infectious disease exists Introduction of a new arbovirus by humans or animals traveling from an endemic area into a susceptible human population in whom the disease is not endemic, or When host susceptibility and response are modified by immunosuppression Dr.K.Prasanthi MD 9

Objectives of an outbreak investigation:

Objectives of an outbreak investigation Identify the responsible etiologic agent . Find the source of infection by studying the occurrence of the disease among persons or in a place or time, as well as determining specific attack rates. Formulate recommendations to prevent further transmission Dr.K.Prasanthi MD 10

Responsibilities of Microbiology lab :

Responsibilities of Microbiology lab Rapid identification of the cause of outbreaks Confirming the diagnosis of disease outbreak Characterization of the infectious agents responsible Tracing the source of infection Detecting the Carriers and Treatment monitoring Dr.K.Prasanthi MD 11

An integrated disease surveillance system :

An integrated disease surveillance system Surveillance involves the scrutiny of all aspects of the occurrence and spread of a disease so that one can bring about effective control Laboratory confirmation : Provides scientific answers to basic epidemiological questions Dr.K.Prasanthi MD 12

Role of Microbiologist:

Role of Microbiologist Ongoing process Passive surveillance of routine diseases and give early confirmation or Active surveillance in the case of out breaks and Epidemiological disease surveillance Dr.K.Prasanthi MD 13

Planning an investigation:

Planning an investigation The primary step for a microbiologist in planning an investigation of an outbreak is to establish a goal to identify the causative agent as in arboviral outbreaks The entire investigation, planning, gathering of data and sample collection must be designed to minimize economic burden Dr.K.Prasanthi MD 14

Syndromic approach :

Syndromic approach Syndromic approach is followed for screening the cases for investigation of an arboviral outbreak Tests has to be done to identify the pathogen for : Acute hemorrhagic fever syndromes by screening for Dengue, hanta virus, KFD, West Nile, malaria, Acute neurological syndrome for AFP, JE, Rabies, and Acute systemic syndrome for Typhoid ,malaria, viral hepatitis, dengue, leptospirosis ,Chikungunya etc Dr.K.Prasanthi MD 15

Broad categories of presentation:

Broad categories of presentation fever Less than seven days duration without any localizing signs ,With rash , With altered sensorium, Bleeding from skin or mucous membrane or fever for more than seven days with or without localizing signs, rash, arthritis or any unusual events causing death or hospitalization Dr.K.Prasanthi MD 16

Critical elements of Laboratory support:

Critical elements of Laboratory support Communication Specimen collection and transport and Specimen processing Dr.K.Prasanthi MD 17

Establishing a Lab Network A Necessary Step:

Establishing a Lab Network A Necessary Step Dr.K.Prasanthi MD 18


Communication A two way communication must be established between the outbreak investigation team and the laboratory Dr.K.Prasanthi MD 19


Communication The laboratory must be informed when there is a suspected outbreak and the nature of that outbreak There must be effective inter sectorial communications between different laboratories where veterinary / entomology investigations are handled , which provides the information about nature of the vector and the possible virus isolated from the vector body In turn the lab must communicate the results of the investigation promptly and accurately to the out break investigation team Dr.K.Prasanthi MD 20

Collection “quality begins with the specimen” :

Collection “ quality begins with the specimen ” Laboratory must be provided with The right specimen Taken at right time Stored & transported in the right way Arrange for an outbreak investigation kit with all the required material Follow the operational guide lines provided during collection of clinical samples Ensure that attending medical staff is knowledgeable / communicated about various aspects of sample collection Dr.K.Prasanthi MD 21

Outbreak investigation kit:

Outbreak investigation kit Disposable storage vials (5ml) Disposable sample collection vials Stool culture bottle Throat swabs blood culture bottles viral transport medium Cary Blair medium Stuart's transport medium Tourniquet , Gloves , Masks Disposable gowns Puncture proof discarding bags (disposable) Vacutainer (plain and EDTA) Syringes and needles Spirit swabs , alcohol swabs Band-aid Vaccine carrier with ice-packs Spirit lamp, Match-box Test tube rack, Centrifuge tubes Lancets Slides and cover slips Rubber bands Ziploc plastic bags Absorbent material (tissue paper, cotton wool, newspaper) Dr.K.Prasanthi MD 22

Required Specimens:

Required Specimens Specimens usually required in a suspected Arboviral outbreak are : Whole Blood Serum CSF Post mortem samples like tissues Dr.K.Prasanthi MD 23


CSF Collected following all aseptic precautions Divide into 3-4 portions 1 st Bottle : Biochemical Analysis 2 nd bottle : Gram staining and culture 3 rd Bottle : cell count etc 4 th Bottle : Serology and virus isolation Do not dilute CSF in VTM Transport with out delay Sent on ice or cold pack insulated containers Dr.K.Prasanthi MD 24

Serum :

Serum Collected in a sterile test tube Allowed to clot Centrifuged and serum separated Transported at 4-8 0 C ( upto 10 days) Paired sera are better 1st sample to be collected during acute stage 2nd sample - After 2-3 weeks Dr.K.Prasanthi MD 25

Blood samples :

Blood samples For Isolation of virus from blood Lymphocytes & PMN’s are the Commonest intracellular sites for virus multiplication Collected following universal precautions Discard the needles in sharp containers Decontaminate the used syringes Dr.K.Prasanthi MD 26

Post mortem samples :

Post mortem samples Biopsy material for relevant tissues Placed in formalin for : HPE Transport medium for microbiological testing sterile saline or viral transport media Specimens in transport media may be transported within 24 hrs at ambient temp Specimens in saline must be transported at 4 - 8 0 C in 48 hours. Dr.K.Prasanthi MD 27

Lab form and Labeling samples:

Lab form and Labeling samples The lab form should be filled and accompany every sample collected Each patient is given an unique ID number Proper labeling of sample : very important Dr.K.Prasanthi MD 28

Storage of the samples:

Storage of the samples Samples should reach the concern lab as early as possible Storage is required when the Laboratory is not in accessible distance , till the collecting boy picks the samples Before storing check again whether all the containers are labeled or not Method of storage for short term storage- refrigerate/ melting ice at 4 0 c delay for more than 48hrs  Freeze at ‘ – 20 0 C’ Dr.K.Prasanthi MD 29

Transportation :

Transportation Transported to identified laboratory The samples should be labeled properly Accompanied with lab form With demographic and epidemiological data Transport boxes tightly packed Absorbent cotton pads in interior Cold chain maintained Avoid large number of samples in single bag Dr.K.Prasanthi MD 30

Laboratory Workup:

Laboratory Workup Lab form and specimen label verified on receipt Ensure that in the laboratory : Equipment and reagents required are available and functional Appropriate safety precautions in place Properly trained staff Quality Control Programme in operation Specimen log maintained Communication abilities Access to electronic communication networks for rapid transmission of results and/ or early warning signals Dr.K.Prasanthi MD 31

Methods of viral diagnosis:

Methods of viral diagnosis Microscopy : Electron Microscope Cell cultures for virus isolation Serology Molecular techniques Dr.K.Prasanthi MD 32

Electron Microscope:

Electron Microscope Rapid identification of viruses especially for detection of fastidious & uncultivable viruses Always exclude bacterial , fungal &parasitic etiology by wet mount, Gram’s stain, negative stain and acid fast stain Immune Electron Microscopy : more Sensitivity and specificity Drawbacks : expensive equipment , difficult to maintain , require experienced observer and sensitivity is often low Dr.K.Prasanthi MD 33

Cell cultures :

Cell cultures Gold standard for virus detection Success of cell cultures depends on the selection of cell lines appropriate specimen maintenance of viability & health of inoculated cells expertness in testing for presence of CPE& haemagglutination activity Dr.K.Prasanthi MD 34

Serology :

Serology Most widely used method for detection of arboviral infections Criteria for diagnosing primary Infection include presence of IgM A single high titer of IgG (or total antibody) though helpful but very unreliable A four fold or more increase in titer of IgG or total antibody between acute and convalescent sera is significant False negatives are seen if the patient is in seroconversion period False positives due to cross reactivity of the closely related species Dr.K.Prasanthi MD 35

Serology :

Serology Criteria for diagnosing reinfection is Four fold or more increase in titer of IgG or total antibody between acute and convalescent sera but absence or slight increase in IgM makes the diagnosis difficult Problems with Serology The time between paired acute and convalescent sera Presence of extensive antigenic cross-reactivity Insignificant titers among Immuno compromised patients Patients given blood or blood products : False Positive Dr.K.Prasanthi MD 36

Serology :

Serology Rapid serologic assays such as IgM-capture ELISA (MAC-ELISA) and IgG ELISA can be employed soon after infection. Early in infection, IgM antibody is more specific, while later in infection, IgG antibody is more reactive. Inclusion of monoclonal antibodies (MAbs ) with defined virus specificities in these solid phase assays has allowed for a level of standardization of Molecular amplification techniques Dr.K.Prasanthi MD 37

Molecular techniques : PCR:

Molecular techniques : PCR Direct impact on rapid response and infectious disease surveillance Essential for the identification of emerging pathogens and for understanding their population structure Useful for characterizing virulence determinants Essential tools for tracking the spread of microbial pathogens May not be possible at district level Identify a nearby lab for doing these tests Dr.K.Prasanthi MD 38

Interpretation The essence of reporting :

Interpretation The essence of reporting Inform the physician of all the positive results If preliminary tests are negative – discuss the diagnostic & therapeutic possibilities with the clinician Discuss with the epidemiologist and veterinarians about the lab data of isolating similar virus from animals and mosquitoes Printed preliminary report should be issued Final report : a legal document ,should be accurate, legible with a definitive identification issued later Reports should be informed immediately to the district health authorities and state health authorities through quick mode of communication Dr.K.Prasanthi MD 39

Interpretation The essence of reporting :

Interpretation The essence of reporting Analyze the locally generated data Interact with other laboratories and epidemiologists Microbiology labs provide an example of effective feedback, achieved by the participation and integration of professionals Dr.K.Prasanthi MD 40


Conclusion Infectious disease surveillance requires the active participation of microbiology laboratories, in which new methodologies and robust information technologies should be implemented in order to guarantee early detection of Arboviral outbreaks. Early response strategies should be designed with the cooperation of microbiology laboratories, in which the efforts of clinical and research microbiologists should be coordinated Dr.K.Prasanthi MD 41

Integrated Effort Can Work Wonders Dr.Prasanthi. K

Integrated Effort Can Work Wonders Dr.Prasanthi. K Dr.K.Prasanthi MD 42

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