ARBOVIRAL  OUTBREAK  A  DEADLY SCOURGE   Role of the Microbiologists in the investigation of an outbreak Dr K.Prasanthi , ...
Introduction <ul><li>Arboviruses  contributing to a large proportion  of total burden of infections </li></ul><ul><li>Viru...
Perspectives  <ul><li>Arboviruses :  </li></ul><ul><ul><li>large group (more than 400) of enveloped RNA viruses which are ...
What is an Outbreak <ul><li>Appearance  of an unusual number of cases of a disease or condition in a population in a given...
INDIAN SCENARIO <ul><li>An epidemic of viral encephalitis was reported in 2005 in India. Where 5,737 persons were affected...
Preparedness:  An Essential Element <ul><li>Because  widespread epizootic activity  , larger outbreaks  of arboviral infec...
Preparedness Is Essential <ul><li>Integrated and accelerated action  to reduce  mortality by these threatening diseases is...
<ul><li>Although clinical diagnosis is  useful,  Laboratory confirmation will be more meaningful  . </li></ul><ul><li>Rapi...
Causes of Arboviral epidemics <ul><li>Travel  of susceptible individuals  into an endemic area where the infectious diseas...
Objectives of an outbreak investigation <ul><li>Identify the responsible etiologic agent . </li></ul><ul><li>Find the sour...
Responsibilities of Microbiology lab  <ul><li>Rapid  identification of the cause  of outbreaks  </li></ul><ul><li>Confirmi...
An integrated disease surveillance  system  <ul><li>Surveillance involves the  scrutiny of all aspects of the occurrence a...
Role of Microbiologist <ul><li>Ongoing process </li></ul><ul><li>Passive  surveillance   </li></ul><ul><ul><li>of  routine...
Planning an investigation <ul><li>The primary step for a microbiologist in planning an investigation of an outbreak is </l...
Syndromic approach   <ul><li>Syndromic approach is followed for screening  the cases for investigation of an arboviral  ou...
Broad  categories of presentation <ul><li>fever  Less than seven days duration without any localizing signs ,With rash , W...
Critical elements of Laboratory  support <ul><li>Communication  </li></ul><ul><li>Specimen collection and transport  and  ...
Establishing a Lab Network A Necessary Step Dr.K.Prasanthi MD LAB  NETWORK PERIPHERAL LABORATORIES INTERMEDIATE LABORATORI...
Communication <ul><li>A two way communication must be established between the outbreak investigation team and the laborato...
Communication <ul><li>The  laboratory must be informed  when there is a suspected outbreak and the nature of that outbreak...
Collection “ quality begins with the specimen ”   <ul><li>Laboratory must be provided with  </li></ul><ul><ul><li>The  rig...
Outbreak  investigation kit <ul><li>Disposable storage vials (5ml) </li></ul><ul><li>Disposable sample collection vials </...
Required Specimens <ul><li>Specimens   usually required in a suspected Arboviral outbreak are : </li></ul><ul><ul><li>Whol...
CSF <ul><li>Collected following all aseptic precautions </li></ul><ul><li>Divide into 3-4 portions </li></ul><ul><ul><li>1...
Serum  <ul><li>Collected in a sterile test tube </li></ul><ul><li>Allowed to clot </li></ul><ul><li>Centrifuged and serum ...
Blood  samples   <ul><li>For  Isolation of virus from blood  </li></ul><ul><li>Lymphocytes & PMN’s   are the Commonest int...
Post mortem samples   <ul><li>Biopsy material for relevant tissues </li></ul><ul><ul><li>Placed in formalin for : HPE </li...
Lab form and Labeling samples <ul><li>The lab form should be filled and accompany every sample collected  </li></ul><ul><l...
Storage of the samples <ul><li>Samples  should reach the concern lab  as early as possible </li></ul><ul><li>Storage is re...
Transportation   <ul><li>Transported to  identified laboratory </li></ul><ul><li>The samples should be labeled properly  <...
Laboratory Workup <ul><li>Lab form and specimen label verified on receipt </li></ul><ul><li>Ensure that in the laboratory ...
Methods of viral diagnosis <ul><li>Microscopy :  Electron Microscope   </li></ul><ul><li>Cell cultures for virus  isolatio...
Electron Microscope <ul><li>Rapid  identification  of viruses especially  for detection of fastidious & uncultivable virus...
Cell cultures   <ul><li>Gold standard  for virus detection  </li></ul><ul><li>Success of cell cultures depends on  the  </...
Serology   <ul><li>Most widely used method for detection of arboviral infections </li></ul><ul><li>Criteria for diagnosing...
Serology   <ul><li>Criteria for diagnosing reinfection  is  </li></ul><ul><ul><li>Four fold or more increase in titer  of ...
Serology   <ul><li>Rapid serologic assays such as  IgM-capture ELISA (MAC-ELISA) and IgG ELISA  can be employed soon after...
Molecular techniques  : PCR <ul><li>Direct  impact on rapid response and infectious disease surveillance  </li></ul><ul><l...
Interpretation  The  essence of reporting  <ul><li>Inform the physician of all the positive results </li></ul><ul><li>If p...
Interpretation  The  essence of reporting  <ul><li>Analyze the locally generated data </li></ul><ul><li>Interact with othe...
Conclusion <ul><li>Infectious  disease surveillance requires the active participation of microbiology laboratories, in whi...
Integrated Effort Can Work Wonders Dr.Prasanthi. K [email_address] Dr.K.Prasanthi MD
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Arboviral outbreak

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Arboviral outbreak

  1. 1. 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
  2. 2. Introduction <ul><li>Arboviruses contributing to a large proportion of total burden of infections </li></ul><ul><li>Viruses are entering into new territories , are more virulent than earlier and becoming endemic in new regions </li></ul><ul><ul><li>Ex :Chikungunya ,Dengue , West nile, JE , KFD, Chandipura which were almost not existing in India , but now reported from all parts of the country </li></ul></ul><ul><li>Severity of these illnesses is totally depends on the efficacy of the surveillance system </li></ul>Dr.K.Prasanthi MD
  3. 3. Perspectives <ul><li>Arboviruses : </li></ul><ul><ul><li>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) </li></ul></ul><ul><li>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 </li></ul><ul><li>Infection is often in apparent or trivial </li></ul><ul><ul><li>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 </li></ul></ul>Dr.K.Prasanthi MD
  4. 4. What is an Outbreak <ul><li>Appearance of an unusual number of cases of a disease or condition in a population in a given period of time and place </li></ul><ul><ul><li>Outbreaks of Japanese Encephalitis were reported largely from India and Nepal </li></ul></ul><ul><ul><li>There were also several outbreaks of Dengue, Chikungunya and JE in recent years </li></ul></ul><ul><ul><ul><li>Resulted in considerable morbidity and mortality </li></ul></ul></ul>Dr.K.Prasanthi MD
  5. 5. INDIAN SCENARIO <ul><li>An epidemic of viral encephalitis was reported in 2005 in India. Where 5,737 persons were affected and 1,344 persons died. </li></ul><ul><li>More than 60 outbreaks of Dengue have occurred since 1956 </li></ul><ul><li>There was a huge outbreak of Dengue in 2003 with 12,754 cases and 215 deaths </li></ul><ul><li>In 2006 outbreak it was estimated that 10,344 cases and 162 deaths due to severe forms of Dengue (DSS/DHF) had taken place. </li></ul><ul><li>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 </li></ul><ul><li>After 32 years in 2005 the disease has reemerged causing 1.3 million cases in 13 states of India including Andhra Pradesh </li></ul>Dr.K.Prasanthi MD
  6. 6. Preparedness: An Essential Element <ul><li>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 </li></ul><ul><li>Private practitioners , the major service providers in India and a frontline to control many diseases are failing to guide people to take preventive measures </li></ul>Dr.K.Prasanthi MD
  7. 7. Preparedness Is Essential <ul><li>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 </li></ul><ul><li>The involvement and expertise of infectious disease physicians, microbiologists, and public health practitioners are essential to the early detection and management of epidemics </li></ul>Dr.K.Prasanthi MD
  8. 8. <ul><li>Although clinical diagnosis is useful, Laboratory confirmation will be more meaningful . </li></ul><ul><li>Rapid alert to outbreak situations may be accelerated with microbiology laboratories integrated into national and supra-national global surveillance programmes </li></ul>Dr.K.Prasanthi MD
  9. 9. Causes of Arboviral epidemics <ul><li>Travel of susceptible individuals into an endemic area where the infectious disease exists </li></ul><ul><li>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 </li></ul><ul><li>When host susceptibility and response are modified by immunosuppression </li></ul>Dr.K.Prasanthi MD
  10. 10. Objectives of an outbreak investigation <ul><li>Identify the responsible etiologic agent . </li></ul><ul><li>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. </li></ul><ul><li>Formulate recommendations to prevent further transmission </li></ul>Dr.K.Prasanthi MD
  11. 11. Responsibilities of Microbiology lab <ul><li>Rapid identification of the cause of outbreaks </li></ul><ul><li>Confirming the diagnosis of disease outbreak </li></ul><ul><li>Characterization of the infectious agents responsible </li></ul><ul><li>Tracing the source of infection </li></ul><ul><li>Detecting the Carriers and </li></ul><ul><li>Treatment monitoring </li></ul>Dr.K.Prasanthi MD
  12. 12. An integrated disease surveillance system <ul><li>Surveillance involves the scrutiny of all aspects of the occurrence and spread of a disease so that one can bring about effective control </li></ul><ul><li>Laboratory confirmation : </li></ul><ul><ul><li>Provides scientific answers to basic epidemiological questions </li></ul></ul>Dr.K.Prasanthi MD
  13. 13. Role of Microbiologist <ul><li>Ongoing process </li></ul><ul><li>Passive surveillance </li></ul><ul><ul><li>of routine diseases and give early confirmation or </li></ul></ul><ul><li>Active surveillance </li></ul><ul><ul><li>in the case of out breaks and </li></ul></ul><ul><li>Epidemiological disease surveillance </li></ul>Dr.K.Prasanthi MD
  14. 14. Planning an investigation <ul><li>The primary step for a microbiologist in planning an investigation of an outbreak is </li></ul><ul><ul><li>to establish a goal </li></ul></ul><ul><ul><li>to identify the causative agent as in arboviral outbreaks </li></ul></ul><ul><li>The entire investigation, planning, gathering of data and sample collection must be designed to minimize economic burden </li></ul>Dr.K.Prasanthi MD
  15. 15. Syndromic approach <ul><li>Syndromic approach is followed for screening the cases for investigation of an arboviral outbreak </li></ul><ul><li>Tests has to be done to identify the pathogen for : </li></ul><ul><ul><li>Acute hemorrhagic fever syndromes by screening for Dengue, hanta virus, KFD, West Nile, malaria, </li></ul></ul><ul><ul><li>Acute neurological syndrome for AFP, JE, Rabies, and </li></ul></ul><ul><ul><li>Acute systemic syndrome for Typhoid ,malaria, viral hepatitis, dengue, leptospirosis ,Chikungunya etc </li></ul></ul>Dr.K.Prasanthi MD
  16. 16. Broad categories of presentation <ul><li>fever Less than seven days duration without any localizing signs ,With rash , With altered sensorium, Bleeding from skin or mucous membrane or </li></ul><ul><li>fever for more than seven days with or without localizing signs, rash, arthritis or any unusual events causing death or hospitalization </li></ul>Dr.K.Prasanthi MD
  17. 17. Critical elements of Laboratory support <ul><li>Communication </li></ul><ul><li>Specimen collection and transport and </li></ul><ul><li>Specimen processing </li></ul>Dr.K.Prasanthi MD
  18. 18. Establishing a Lab Network A Necessary Step Dr.K.Prasanthi MD LAB NETWORK PERIPHERAL LABORATORIES INTERMEDIATE LABORATORIES NATIONAL REFRENCE CENTRES
  19. 19. Communication <ul><li>A two way communication must be established between the outbreak investigation team and the laboratory </li></ul>Dr.K.Prasanthi MD
  20. 20. Communication <ul><li>The laboratory must be informed when there is a suspected outbreak and the nature of that outbreak </li></ul><ul><li>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 </li></ul><ul><li>In turn the lab must communicate the results of the investigation promptly and accurately to the out break investigation team </li></ul>Dr.K.Prasanthi MD
  21. 21. Collection “ quality begins with the specimen ” <ul><li>Laboratory must be provided with </li></ul><ul><ul><li>The right specimen </li></ul></ul><ul><ul><li>Taken at right time </li></ul></ul><ul><ul><li>Stored & transported in the right way </li></ul></ul><ul><li>Arrange for an outbreak investigation kit with all the required material </li></ul><ul><li>Follow the operational guide lines provided during collection of clinical samples </li></ul><ul><li>Ensure that attending medical staff is knowledgeable / communicated about various aspects of sample collection </li></ul>Dr.K.Prasanthi MD
  22. 22. Outbreak investigation kit <ul><li>Disposable storage vials (5ml) </li></ul><ul><li>Disposable sample collection vials </li></ul><ul><li>Stool culture bottle </li></ul><ul><li>Throat swabs </li></ul><ul><li>blood culture bottles </li></ul><ul><li>viral transport medium </li></ul><ul><li>Cary Blair medium </li></ul><ul><li>Stuart's transport medium </li></ul><ul><li>Tourniquet , Gloves , Masks </li></ul><ul><li>Disposable gowns </li></ul><ul><li>Puncture proof discarding bags (disposable) </li></ul><ul><li>Vacutainer (plain and EDTA) </li></ul><ul><li>Syringes and needles </li></ul><ul><li>Spirit swabs , alcohol swabs </li></ul><ul><li>Band-aid </li></ul><ul><li>Vaccine carrier with ice-packs </li></ul><ul><li>Spirit lamp, Match-box </li></ul><ul><li>Test tube rack, Centrifuge tubes </li></ul><ul><li>Lancets </li></ul><ul><li>Slides and cover slips </li></ul><ul><li>Rubber bands </li></ul><ul><li>Ziploc plastic bags </li></ul><ul><li>Absorbent material (tissue paper, cotton wool, newspaper) </li></ul>Dr.K.Prasanthi MD
  23. 23. Required Specimens <ul><li>Specimens usually required in a suspected Arboviral outbreak are : </li></ul><ul><ul><li>Whole Blood </li></ul></ul><ul><ul><li>Serum </li></ul></ul><ul><ul><li>CSF </li></ul></ul><ul><ul><li>Post mortem samples like tissues </li></ul></ul>Dr.K.Prasanthi MD
  24. 24. CSF <ul><li>Collected following all aseptic precautions </li></ul><ul><li>Divide into 3-4 portions </li></ul><ul><ul><li>1 st Bottle : Biochemical Analysis </li></ul></ul><ul><ul><li>2 nd bottle : Gram staining and culture </li></ul></ul><ul><ul><li>3 rd Bottle : cell count etc </li></ul></ul><ul><ul><li>4 th Bottle : Serology and virus isolation </li></ul></ul><ul><li>Do not dilute CSF in VTM </li></ul><ul><li>Transport with out delay </li></ul><ul><ul><li>Sent on ice or cold pack insulated containers </li></ul></ul>Dr.K.Prasanthi MD
  25. 25. Serum <ul><li>Collected in a sterile test tube </li></ul><ul><li>Allowed to clot </li></ul><ul><li>Centrifuged and serum separated </li></ul><ul><li>Transported at 4-8 0 C ( upto 10 days) </li></ul><ul><li>Paired sera are better </li></ul><ul><ul><li>1st sample to be collected during acute stage </li></ul></ul><ul><ul><li>2nd sample - After 2-3 weeks </li></ul></ul>Dr.K.Prasanthi MD
  26. 26. Blood samples <ul><li>For Isolation of virus from blood </li></ul><ul><li>Lymphocytes & PMN’s are the Commonest intracellular sites for virus multiplication </li></ul><ul><li>Collected following universal precautions </li></ul><ul><li>Discard the needles in sharp containers </li></ul><ul><li>Decontaminate the used syringes </li></ul>Dr.K.Prasanthi MD
  27. 27. Post mortem samples <ul><li>Biopsy material for relevant tissues </li></ul><ul><ul><li>Placed in formalin for : HPE </li></ul></ul><ul><ul><li>Transport medium for microbiological testing </li></ul></ul><ul><ul><ul><li>sterile saline or viral transport media </li></ul></ul></ul><ul><li>Specimens in transport media may be transported within 24 hrs at ambient temp </li></ul><ul><li>Specimens in saline must be transported at 4 - 8 0 C in 48 hours. </li></ul>Dr.K.Prasanthi MD
  28. 28. Lab form and Labeling samples <ul><li>The lab form should be filled and accompany every sample collected </li></ul><ul><li>Each patient is given an unique ID number </li></ul><ul><li>Proper labeling of sample : very important </li></ul>Dr.K.Prasanthi MD
  29. 29. Storage of the samples <ul><li>Samples should reach the concern lab as early as possible </li></ul><ul><li>Storage is required when the Laboratory is not in accessible distance , till the collecting boy picks the samples </li></ul><ul><li>Before storing check again whether all the containers are labeled or not </li></ul><ul><li>Method of storage </li></ul><ul><ul><li>for short term storage- refrigerate/ melting ice at 4 0 c </li></ul></ul><ul><ul><li>delay for more than 48hrs  Freeze at ‘ – 20 0 C’ </li></ul></ul>Dr.K.Prasanthi MD
  30. 30. Transportation <ul><li>Transported to identified laboratory </li></ul><ul><li>The samples should be labeled properly </li></ul><ul><li>Accompanied with lab form </li></ul><ul><ul><li>With demographic and epidemiological data </li></ul></ul><ul><li>Transport boxes tightly packed </li></ul><ul><ul><li>Absorbent cotton pads in interior </li></ul></ul><ul><li>Cold chain maintained </li></ul><ul><li>Avoid large number of samples in single bag </li></ul>Dr.K.Prasanthi MD
  31. 31. Laboratory Workup <ul><li>Lab form and specimen label verified on receipt </li></ul><ul><li>Ensure that in the laboratory : </li></ul><ul><ul><li>Equipment and reagents required are available and functional </li></ul></ul><ul><ul><li>Appropriate safety precautions in place </li></ul></ul><ul><ul><li>Properly trained staff </li></ul></ul><ul><ul><li>Quality Control Programme in operation </li></ul></ul><ul><ul><li>Specimen log maintained </li></ul></ul><ul><ul><li>Communication abilities </li></ul></ul><ul><li>Access to electronic communication networks for rapid transmission of results and/ or early warning signals </li></ul>Dr.K.Prasanthi MD
  32. 32. Methods of viral diagnosis <ul><li>Microscopy : Electron Microscope </li></ul><ul><li>Cell cultures for virus isolation </li></ul><ul><li>Serology </li></ul><ul><li>Molecular techniques </li></ul>Dr.K.Prasanthi MD
  33. 33. Electron Microscope <ul><li>Rapid identification of viruses especially for detection of fastidious & uncultivable viruses </li></ul><ul><li>Always exclude bacterial , fungal &parasitic etiology by wet mount, Gram’s stain, negative stain and acid fast stain </li></ul><ul><li>Immune Electron Microscopy : more Sensitivity and specificity </li></ul><ul><li>Drawbacks : expensive equipment , difficult to maintain , require experienced observer and sensitivity is often low </li></ul>Dr.K.Prasanthi MD
  34. 34. Cell cultures <ul><li>Gold standard for virus detection </li></ul><ul><li>Success of cell cultures depends on the </li></ul><ul><ul><li>selection of cell lines </li></ul></ul><ul><ul><li>appropriate specimen </li></ul></ul><ul><ul><li>maintenance of viability & health of inoculated cells </li></ul></ul><ul><ul><li>expertness in testing for presence of CPE& haemagglutination activity </li></ul></ul>Dr.K.Prasanthi MD
  35. 35. Serology <ul><li>Most widely used method for detection of arboviral infections </li></ul><ul><li>Criteria for diagnosing primary Infection include </li></ul><ul><ul><li>presence of IgM </li></ul></ul><ul><ul><li>A single high titer of IgG (or total antibody) though helpful but very unreliable </li></ul></ul><ul><ul><li>A four fold or more increase in titer of IgG or total antibody between acute and convalescent sera is significant </li></ul></ul><ul><li>False negatives are seen if the patient is in seroconversion period </li></ul><ul><li>False positives due to cross reactivity of the closely related species </li></ul>Dr.K.Prasanthi MD
  36. 36. Serology <ul><li>Criteria for diagnosing reinfection is </li></ul><ul><ul><li>Four fold or more increase in titer of IgG or total antibody between acute and convalescent sera </li></ul></ul><ul><ul><li>but absence or slight increase in IgM makes the diagnosis difficult </li></ul></ul><ul><li>Problems with Serology </li></ul><ul><ul><li>The time between paired acute and convalescent sera </li></ul></ul><ul><ul><li>Presence of extensive antigenic cross-reactivity </li></ul></ul><ul><ul><li>Insignificant titers among Immuno compromised patients </li></ul></ul><ul><ul><li>Patients given blood or blood products : False Positive </li></ul></ul>Dr.K.Prasanthi MD
  37. 37. Serology <ul><li>Rapid serologic assays such as IgM-capture ELISA (MAC-ELISA) and IgG ELISA can be employed soon after infection. </li></ul><ul><li>Early in infection, IgM antibody is more specific, while later in infection, IgG antibody is more reactive. </li></ul><ul><li>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 </li></ul>Dr.K.Prasanthi MD
  38. 38. Molecular techniques : PCR <ul><li>Direct impact on rapid response and infectious disease surveillance </li></ul><ul><li>Essential for the identification of emerging pathogens and for understanding their population structure </li></ul><ul><li>Useful for characterizing virulence determinants </li></ul><ul><li>Essential tools for tracking the spread of microbial pathogens </li></ul><ul><li>May not be possible at district level </li></ul><ul><ul><li>Identify a nearby lab for doing these tests </li></ul></ul>Dr.K.Prasanthi MD
  39. 39. Interpretation The essence of reporting <ul><li>Inform the physician of all the positive results </li></ul><ul><li>If preliminary tests are negative – discuss the diagnostic & therapeutic possibilities with the clinician </li></ul><ul><li>Discuss with the epidemiologist and veterinarians about the lab data of isolating similar virus from animals and mosquitoes </li></ul><ul><li>Printed preliminary report should be issued </li></ul><ul><li>Final report : a legal document ,should be accurate, legible with a definitive identification issued later </li></ul><ul><li>Reports should be informed immediately to the district health authorities and state health authorities through quick mode of communication </li></ul>Dr.K.Prasanthi MD
  40. 40. Interpretation The essence of reporting <ul><li>Analyze the locally generated data </li></ul><ul><li>Interact with other laboratories and epidemiologists </li></ul><ul><li>Microbiology labs provide an example of effective feedback, achieved by the participation and integration of professionals </li></ul>Dr.K.Prasanthi MD
  41. 41. Conclusion <ul><li>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. </li></ul><ul><li>Early response strategies should be designed with the cooperation of microbiology laboratories, in which the efforts of clinical and research microbiologists should be coordinated </li></ul>Dr.K.Prasanthi MD
  42. 42. Integrated Effort Can Work Wonders Dr.Prasanthi. K [email_address] Dr.K.Prasanthi MD

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