Case study: Investigation of an outbreak of



Case study

An outbreak with respiratory symptoms

Toronto, Canada

This case study was developed by a working group led by WHO EPR. It is based on the investigation of an outbreak of acute respiratory symptoms in 2003 in Toronto, Canada. The data have been modified for the purposes of the case study.

Using this case study in the classroom:

We recommend that this case study be used in conjunction with the “Laboratory Skills for Epidemiologists” module, developed by the WHO. However, it can be delivered on its own, provided key lectures are presented first.

Recommended prerequisite lectures (cf. training matrix):

Lecture 6: Principles of immunology

Lecture 7: Antibody and antigen detection

Lecture 10: Viral culture

Lecture 3: Taking appropriate and adequate samples safely

Lecture 4:Transport, disinfection and biosafety

Lecture 13: Sequencing and phylogeny (if Part 10 is to be done)

Time required for this case study:

3hours for each session

This case study does not come with a facilitator’s guide. The answers to all the questions for each section are provided as an introduction to the following section.

To run this case study in the classroom, we propose that it be distributed one page at a time. Participants should take turns reading it aloud, paragraph by paragraph. Reading all paragraphs aloud and in turns has two advantages: first, everyone can quickly participate and get beyond the inhibition of having her/his voice heard in a large room; second, time is given to the whole class to understand the issue and think about the answers. The participants reading the question may try to answer it if s/he can; otherwise, it can be discussed as a group. The next participant reads the next question and so on until the end of the page. After the next part/page is distributed, the next participant continues and so on until the case study is over. Once the epilogue is read, re-visit the objectives – this reinforces the learning and provides an opportunity to clarify any remaining issues.

Learning objectives

1. Understand the importance of laboratory data for surveillance of respiratory pathogens and outbreak detection;

2. Understand the limitations of laboratory data in surveillance and outbreak detection;

3. Devise an optimal sampling strategy for the type and the number of samples to take during an outbreak of respiratory illness;

4. Follow ethical guidelines and consider requirements for informed consent in patient sampling;

5. Use personal protective measures to prevent infections when taking samples and preparing them for transport;

6. Transport samples according to national and/or international regulations, as applicable;

7. Identify the laboratory techniques that may be used to test specimens collected during a respiratory outbreak;

8. Understand limitations of results of PCR and serology testing;

9. Interpret phylogenetic trees to understand the spread of disease; origin and relatedness of the pathogen;

10. Interpret laboratory data in context of epidemiological data in order to make recommendations for public health action

Prologue

On February 14th, 2003, the World Health Organization (WHO) reported 305 cases and two deaths of an unknown respiratory disease in Guangdong Province, China.

On February 21st a doctor from that province arrived in Hong Kong and stayed at the Metropole Hotel, unaware that he had been exposed to this unknown, respiratory pathogen and was now infected. His illness marked the beginning of a global outbreak. The story continues in Toronto, Canada in late February….

Part A case of acute respiratory illness in Toronto

Between February 18th and 21st, 2003, a 78-year old woman was on vacation in Hong Kong with her husband – they stayed at the Metropole Hotel. Upon her return home to Toronto, Canada, on February 23rd she developed a fever, myalgia, sore throat and a cough. She was cared for by her family at her home. Over the next 10 days, her condition deteriorated and she died at home on March 5th.

On February 27th, this woman’s 41-year old son (Case A) became ill with fever and respiratory symptoms. On March 7th, 2003, he presented to the emergency room (ER) of Hospital A and was admitted to the Intensive Care Unit (ICU) where he died on March 13th.

Between March 3rd and 14th, four more family members developed similar symptoms. The doctor who provided care to this family on March 6th developed respiratory symptoms on March 10th.

By March 19th, several nurses who worked in the ER and ICU of Hospital A reported ill with fever and one or more of these symptoms: cough, malaise, myalgia, and headache. They were told to isolate themselves at home and to wear masks. The local public health department was notified of this hospital cluster.

As a Field Epidemiologist placed with the local public health department, you are called in to assist.

Question 1a

Is this an outbreak?

Question 1b

What source(s) of routine surveillance data could help you confirm the existence of an outbreak in this investigation?

Question 1c

List the types of pathogens (bacterial, viral etc.) that could be considered in the differential diagnosis in an outbreak of respiratory illness.

Question 1d

Who should be on the outbreak team?

Discussion for Part 1

Because we expect respiratory infections to spread within a family, the situation was not initially defined as an outbreak. In Toronto, it was initially thought that the index case and her son (Case A) had tuberculosis (TB). The family members were being investigated as potential TB case contacts. However, the two deaths and the rapid onset of respiratory symptoms among hospital staff raised suspicion that there was something going on unrelated to TB.

The categories of pathogens included in the differential diagnosis for a respiratory infection include: viral, bacterial, fungal and parasitic infections (though unlikely as it would not present like an outbreak). A list of causes of respiratory illness is provided in Appendix 1.

As in any outbreak investigation, you should be part of a multidisciplinary team, including epidemiologists, laboratory specialists and clinicians, if possible with expertise in different content areas.

During respiratory disease outbreaks, laboratory surveillance data related to influenza and other reportable respiratory disease are useful - in reviewing these, epidemiologists can verify baseline (expected) case counts, and may be able to identify unusual patterns/activity – leading or helping confirm the existence of an outbreak. Systematic laboratory testing to identify the pathogen is an important first step in any outbreak. In this scenario, while the causative agent is unknown, supplementary information from the lab can help rule out the differential diagnoses.

Part Collecting laboratory specimens

On March 12th, 2003, the WHO issued an alert about a new illness called Severe Acute Respiratory Syndrome (SARS); global surveillance activities were implemented. Based on the epidemiological and laboratory information available, the Toronto Public Health (TPH) department and clinicians identify the Toronto situation as a SARS outbreak.

Recognizing the need to coordinate laboratory-based surveillance with the epidemiologic investigation, TPH contacts the microbiologist at the provincial laboratory on March 20th to ensure parties keep one another updated.

The outbreak investigation team includes hospital and public health representatives. They decide to look for other cases of SARS and adopt the outbreak case definition set by WHO:

Suspect case: Person of any age with a history of high fever (>38°C) AND cough or breathing difficulty AND one or more of the following exposures during the 10 days prior to onset of symptoms: close contact with a probable case of SARS, history of travel to affected areas (e.g., Hong Kong).

Probable case: A suspect case with radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome on chest x-ray.

As part of your hypothesis generation, you determine that you should interview one of the ill hospital staff members at home. You would collect the epidemiologic information and a nurse will collect specimens for laboratory testing. The nurse seeks your advice in determining what kind of specimens she needs to collect and what she needs to bring along with her.

Question 2a

List the different samples that could be taken during outbreaks of respiratory illness caused by an unknown pathogen.

Question 2b

In this investigation, what kind of samples should be collected? What materials will you need?

Question 2c

1) From whom should you obtain samples?

2) In this situation, would you consider testing i)individuals not meeting the outbreak case definition or ii)healthy contacts of ill individuals? If so, why?

3) How many individuals should you sample? Why?

4) How many samples would you take from each person?

Question 2d

Will you need to use transport media?

If so, what kind of transport media should be used for viral specimens?

Discussion for Part 2

Appendix 2 shows the range of specimens that could be taken during respiratory outbreaks of unknown etiology, and the materials required to obtain them. You consult with the microbiologist at the laboratory about types of samples to collect.

You determine that the nurse should obtain a respiratory sample (a nasopharyngeal (NP) swab). Nasopharyngeal aspirates (NPA) are more invasive, are harder to collect and can produce more contamination – so while they were initially considered, NP swabs were favoured. In addition to the NP swab, a whole blood sample is obtained to look for circulating antigens.

Before heading off to the field, you ask the microbiologist about specimen collection and shipment protocols. She recommends that you transport the NP swab in viral transport media (VTM) to improve virus survival, but notes that such media are not required if cold chain is ensured and if the laboratory can process the specimens promptly. Transport media are also used to maintain viability of bacterial samples – but viability is also dependant on the pathogen.

To transport the NP swab and whole blood sample you ask the nurse to bring:

• a styrofoam container/cooler with ice packs to maintain cold chain for collected samples;

• sterile rayon swabs with plastic shafts for the NP swab;

and

• sterile EDTA specimen tubes for collection of whole blood (lavender top).

Sampling protocol

As the causative pathogen is unknown, the laboratory advises taking samples from all ill individuals. Since multiple tests may be required to exclude all differential organisms, with the potential identification of a new pathogen and the subsequent need to send additional samples to multiple (reference) laboratories, the laboratory requests that, if possible, additional specimens be taken.

The number of samples needed is generally based on:

i) sensitivity of the test

ii) the importance of avoiding misclassification

iii) whether this is a known or unknown pathogen

Serial sampling on consecutive days can increase the probability of identifying an unknown agent and assists in determining the natural course of the unknown disease.

While laboratory testing is usually done to test for a causative agent based on a predetermined hypothesis, in this outbreak, testing of contacts who did not meet the case definition is considered because of the potentially high infectious nature and the seriousness of illness. This is especially important early in an outbreak with an unknown pathogen, as the case definition may need to be refined (i.e., too specific).

Other reasons for testing of contacts not meeting the outbreak case definition include:

• Describing the natural history of infection

• Determining whether asymptomatic infection is possible

• Estimating the secondary attack rate

Part Ethical considerations

As described above, specimens were taken from ill and healthy persons and personal information was collected from both groups. This process can be both uncomfortable and invasive.

Question 3a

What are the ethical considerations in testing these contacts?

Question 3b

How do you ensure confidentiality in a high profile outbreak?

Discussion for Part 3

Before collecting epidemiologic information or biological specimens, it is important that people understand why and how they were identified and selected, why their personal information and specimens are required, and how the information generated will be used; data and specimens should only be collected once informed consent is obtained.

Normally, if specimens are collected for purely academic research, approval from an ethics committee and written consent from participant’s and assurances of confidentiality are absolutely necessary; this is usually time-consuming and can add significant delay to such studies. Outbreak investigations are usually exempt from ethics committee clearance, and can usually proceed under public health legislation. Nonetheless, good practice (respecting confidentiality), provision of informed consent (written if possible) and self-imposed ethical standards must be maintained during outbreaks.

Because this is an outbreak of a new communicable disease, you and the laboratory agree that you need as much information as possible (e.g., to detect subclinical cases) and therefore healthy contacts of cases or mildly symptomatic individuals are asked to provide samples. As lab results became available, some case contacts test positive for the causative agent for SARS. While some of these contacts will eventually meet the WHO case definition for SARS, others remain mildly symptomatic and do not meet the case definition.

While specimens collected for diagnostic purposes should be labelled with the patient’s name, you point out to the team that it is important to not generate or release any data that could potentially identify individuals. The team agrees to avoid publishing patient names or personal identifiers in reports.

Part Taking the sample

The nurse is preparing to collect the samples from the case.

Question 4A

What personal precautions should be taken when interacting with possible cases infected with an unknown infectious respiratory pathogen?

Discussion for Part 4

Since the mode of SARS transmission is still unknown, the field team visiting the suspect cases must wear personal protective equipment (PPE) while conducting interviews and when obtaining specimens,

It is likely that the causative agent in the outbreak is a respiratory pathogen. Respiratory pathogens can be spread by respiratory droplets, through direct or indirect contact with the patient, or by the airborne route, particularly if the individual coughs or sneezes during an interview or when collecting biologic specimens.

Precautions against all possible modes of transmission are taken (see Table 2); before entering the homes, gloves, gown, eye protection and masks are worn. To be effective, you must ensure that the equipment you are donning fit you properly.

Table 2: Infection control precautions to interrupt transmission of pathogens

|Precautions |Use |Requirements |

|Contact precautions |For patients known or suspected to have serious illnesses easily |Gloves, gown |

| |transmitted by direct patient contact or by contact with items in | |

| |the patient's environment | |

|Droplet precautions |Barrier to stop infections spread by large (>5 microns), moist |Contact precautions + well-fitting mask + |

| |droplets produced by people when they cough, sneeze or speak |eye protection |

|Airborne precautions |For patients known or suspected to have serious illnesses |Contact and droplet precautions + N95 |

| |transmitted by airborne droplet nuclei |mask, isolation room (in hospital) |

Part Laboratory selection

The nurse is preparing and packaging the NP swabs for shipment to the laboratory. You consult with your colleagues as to the appropriate laboratory to send these specimens. Your colleagues offer you several choices (the hospital lab, the regional lab…etc). You vaguely recall a discussion with a microbiologist relating to laboratory designation according to pathogen biosafety levels.

Question 5a

What biosafety level do you think is appropriate for SARS?

Question 5b

What documentation should accompany your team’s samples to ensure timely processing?

Discussion for Part 5

Laboratories designate biosafety precautions for specimens based on the severity of disease and the potential for a cure. In this outbreak, it is important to identify a laboratory capable and designated to handle highly infectious agents and with experience in different methodology. Assistance from international and reference laboratories should also be considered. Appendix 4 references the different laboratory biosafety levels and the work they can carry out.

In this situation with an unknown pathogen, BioSafety Level (BSL)-3 handling should be carried out, since the samples may contain a live virus. This means samples will be handled under a safety hood and/or with PPE plus a full-face shield.

Before collecting the sample, you should consult with the laboratory point of contact, to ensure timely transport and processing of the samples.

The following minimal information needs to accompany the sample:

1. Type of sample

2. Place

3. Symptoms and dates of onset

4. Date of specimen collection

5. Patient Name/unique identifier/outbreak code

Note: Steps should be taken to ensure the link between the patient and specimen is maintained. When there are multiple samples taken from each patient or when several laboratories are involved in testing and use different identifiers, it may be hard to re-match results to patients. As a minimum, use date of birth and sex consistently for each sample taken.

The samples which your team collects are to be sent to the National Microbiology Laboratory (NML) in Winnipeg, Manitoba (a two hour flight away), which includes BSL-3 and 4 facilities. In other countries experiencing SARS, the WHO provides a list of appropriate laboratories where samples could be sent.

--------END OF Session 1-------

Part Transportation of respiratory specimens

Samples from the ill and their contacts have been taken and the appropriate laboratory has been identified and contacted.

On March 21st, the samples are ready to be sent to the national laboratory. The WHO has also requested parallel specimens for testing.

Question 6a

What kind of packaging is necessary to prepare these samples for transport? Consider both national and international shipment requirements.

Question 6b

Would you request a cold chain for the transport of these nasopharyngeal specimens? Why?

Discussion for Part 6

Shipping potentially infectious samples is a highly regulated issue that should only be performed by trained individuals. If regulations are ignored, it can not only lead to heavy fines (and even imprisonment), but the sample may not be accepted by the transporting body, the sample may become destroyed, and in a worst case scenario, the sample could infect people who come into contact with the package. National regulations for the transport of infectious substances exist in most countries. In Canada, the Transportation of Dangerous Goods Act provides procedures for shipping of specimens.

Generally, substances that contain potentially infectious agents should be transported in a “triple package”:

● Primary receptacle*: a leak proof specimen container. In case of breakage, the receptacle is packaged with sufficient absorbent material to absorb its entire contents..

● Secondary packaging*: a leak proof secondary container which encloses and protects the primary receptacle(s). Several cushioned primary receptacles may be placed in one secondary package, but additional absorbent material should be added to the package -- enough to absorb all fluids contained, in case of breakage.

● Outer packaging*: the secondary package(s) are placed in outer shipping packaging with suitable cushioning material. Outer packaging protects the contents from outside influences, such as physical damage, while in transit. The smallest overall external dimension shall be 10x10 cm.

*Please refer to current International Airline and Transport Agency (IATA) shipping guidelines for more details about definitions, packaging requirements, markings and labels, accompanying documentation, and refrigerants.

Example of triple package:

Part Laboratory testing

The outbreak continues over the next few weeks…

On March 24th, 2003, facilitated by international epidemiologic and laboratory collaboration, a new coronavirus is identified from a SARS case. Between April 8th–10th, researchers find positive antibody titers to SARS CoV in a high percentage of SARS patients and no titer in control patients. Finally, on April 16th, the WHO announces that this coronavirus, never before seen in humans, is the causative agent of SARS, based on study results that found that monkeys infected with the pathogen developed similar symptoms to those observed in human SARS cases. The speed with which the causative agent of SARS was identified can be attributed in part through this excellent, and unprecedented, laboratory-epidemiology collaboration between 13 laboratories in 10 countries.

After reviewing the descriptive epidemiology for this outbreak to date, your team finds that contact and droplet precautions implemented in Hospital A reduced the nosocomial spread of SARS. As a result, you decide that the Toronto data support the leading hypothesis that SARS spreads by respiratory droplets.

On March 25, the samples obtained by the nurse that accompanied you reach the national laboratory and are tested for respiratory pathogens and SARS CoV. The laboratory stores parts of the samples under appropriate conditions for further testing.

With no standard test for SARS established yet, what are the potential tests that could be considered?

Question 7a

What types of tests could be used to look for antibodies to the SARS pathogen?

What types of tests could be used to look for the SARS pathogen (coronavirus) itself?

Discussion for Part 7

Table 3: Available tests for detection of SARS-CoV.

|Test Category |Purpose of Test |Comments/Interpretation |

|Direct Tests | | |

|Virus isolation (culture) |To provide evidence that a person has been |A negative test does not disprove this hypothesis; a |

| |infected with the virus. |negative result could be due to timing of specimen |

| | |collection, no live virus in the sample and/or a poor|

| |In the course of the identification of the |handling of the culture or using the wrong cells to |

| |SARS-CoV, it is important to prove that it is also|grow the virus in. |

| |the causative agent. | |

| | |Requires BSL-3 laboratory. |

|Molecular methods (i.e., PCR) |To provide evidence that genetic material of the |A negative test does not rule out virus in the sample|

| |virus is present in the sample. |nor that the patient had the infection. It can be due|

| | |to low sensitivity of the PCR or timing of the sample|

| |Note that PCR, particularly RT-PCR is prone to |collection. |

| |contamination. |A positive test must be interpreted as such only |

| | |after ascertaining that laboratory |

| | |cross-contamination did not occur. |

|Indirect Tests | | |

|Enzyme-linked immunosorbent |To detect a mixture of IgM and IgG antibodies in |IgG usually remains detectable after resolution of |

|assay (ELISA) |the serum of SARS patients. |the illness. Reliably yields positive results at, or|

| | |after day 28 following SARS onset |

| | |Separate IgG/IgM tests were not available at the |

| | |beginning |

|Immunofluorescence |To detect IgM or IgG (or both) in the serum of |Expect positive results at, or after day 28 following|

|assay (IFA) |SARS patients. |SARS onset. Results may be quantified by using serial|

| | |titrations of patient sera. |

| |This technique uses SARS CoV-infected cells fixed | |

| |on a microscope slide. Immunofluorescent-labeled | |

| |secondary antibodies against human IgG and/or IgM | |

| |detect patient antibodies binding to viral | |

| |antigens using an immunofluorescence microscope | |

|Neutralization |To assess (via titration) the ability of patient |Best correlate of immunity. However, must be done in |

|test (NT) |sera to neutralize the infectivity of SARS-CoV on |institutions with BSL-3 facilities and is expensive |

| |cell culture |and time-consuming. |

Direct Tests: demonstration of the presence of the infectious agent/pathogen

Indirect Tests: demonstration of the presence of antibodies to a particular agent

Antibody tests

A positive IgG antibody test result indicates previous infection with SARS-CoV. The microbiologist informs you that seroconversion from negative to positive or a four-fold rise in the antibody titer from acute to convalescent serum indicates a recent infection. A negative antibody test taken more than 28 days after symptom onset is likely to indicate no infection with SARS-CoV.

You and the laboratory discuss that there seems to be no background seroprevalence against SARS-CoV in control populations screened so far. Antibody testing allows the indirect diagnosis of SARS-CoV infection but has the advantage of being independent of the sample type, in contrast to other virus detection methods. Disadvantages include a lack of usefulness in the early stages of SARS (an IgG, not IgM test) and some SARS cases have negative antibody tests. A positive antibody test indicates previous infection with SARS CoV (helpful to confirm the diagnosis) but a negative test in a patient who meets the case definition of SARS does not necessarily disprove the diagnosis.

Molecular tests

To design a PCR probe, parts of the genomic sequence of the pathogen have to be known and must be specific for the virus. The latest tests for SARS used two different regions of the gene (the replicase and nuclease gene) as targets.

There are different methods and names for PCR tests:

1) RT-PCR: Since PCR has to be done with a reverse transcriptase on a RNA-virus

2) Nested and unnested PCRs: Nested PCR means that first a larger section of the gene is replicated and then the specific area is targeted. Nested PCRs are usually more sensitive, but they bear a higher risk for contamination.

3) Real-time PCR: Where each cycle of the PCR is analysed and the amount of the virus in the sample can be estimated.

Part Confirmation of SARS Coronavirus infection

Now that confirmatory tests have been developed to detect SARS-CoV infection, you can include laboratory results in your case definition.

Question 8A

What could be a possible laboratory-confirmed case definition for SARS?

Discussion for Part 8

WHO recommends the following case definition for laboratory-confirmed SARS cases:

A person with clinically-compatible illness and one of:

• Detection of antibody to SARS-CoV in specimens obtained during acute illness or >28 days after illness onset,

OR

• Detection of SARS-CoV RNA by RT-PCR, confirmed by a second PCR assay by using a second aliquot of the specimen and a different set of PCR primers,

OR

• Isolation of SARS CoV

By May 2003, 257 probable and suspect SARS cases associated with the Toronto area outbreak had been reported. Of these, approximately 63% were health-care acquired infections, 32% were household or close contacts of cases and 5% were travel-related (imported cases). Twenty-seven SARS-related deaths were also reported.

Part 9 Interpretation of laboratory results

Available serology and PCR results from those tested for SARS (including both symptomatic and asymptomatic contacts of cases) are now available. A summary is presented in Table 4.

Table 4:

|Specimen |PCR positive |PCR negative |

| |Seropositive |Seronegative |Seropositive |Seronegative |

|EDTA blood |4 |19 |74 |296 |

|Nasopharyngeal swab |21 |29 |48 |320 |

|Throat swab |11 |9 |32 |278 |

|Stool |8 |0 |7 |32 |

|Sputum |6 |2 |0 |9 |

Using convalescent serology as the “gold standard”, the team decides to look at the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR tests as a diagnostic tool.

Question 9a

For the PCR test, calculate and describe the

a) sensitivity

b) specificity

c) positive predictive value

d) negative predictive value

Question 9b

Interpret these values. Which of these do you think is most relevant for public health decision-making?

Question 9c

How do you interpret positive PCR test results for SARS CoV in individuals who do not meet the outbreak case definition?

Question 9d

If there were a case of SARS reported in a setting where there were no known cases (ruling out travel-related importations), how would you interpret a positive PCR test result for SARS CoV?

Question 9e

How do you interpret negative PCR results for SARS CoV in individuals who meet the case definition?

Question 9f

Human metapneumovirus was also identified in some specimens from SARS patients. Discuss possible interpretations of this result.

Answer to Part 9

Answer 9a

| | |Gold standard (Serology) |

| | |+ |- |

|Test |+ |a (true positive) |b (false positive) |

|(PCR) | | | |

| |- |c (false negative) |d (true negative) |

Table 5: Accuracy calculations for PCR tests for SARS-CoV

|Specimen |Sensitivity |Specificity |PPV (%) |NPV (%) |

| |(%) |(%) | | |

|EDTA blood |5 |94 |17 |80 |

|NP swab |30 |92 |42 |87 |

|Throat swab |53 |97 |55 |90 |

|Stool |53 |100 |100 |82 |

|Sputum |100 |82 |75 |100 |

Sensitivity of a test asks “How many true cases were detected using the test?”. For SARS we want a test that is highly sensitive because of the implications of missing a true case (i.e., transmission of illness). Using these data, the test sensitivity was low for the specimens most frequently taken (blood, NP and throat swab) when using the PCR test results used at that time.

Note that the PPV of the PCR depends on the type of PCR (the protocol and primer used) --so the PPV calculated is specific for the PCR used here.

Specificity of a test asks “How many true non-cases will test negative?”. Based on your data, you found that specificity of the PCR tests were high.

Answer 9b

Public health relevance

In order to interpret the data correctly, and especially during outbreaks, clinicians and epidemiologists need to understand the level of confidence that a positive diagnostic test is a true case and not a false positive. This is the PPV of the test. The NPV is the probability that with a negative test, the person is not infected.

The PPV (rate of false positives detected) should be low– the implications of detecting false positives would in this outbreak would lead to unintentional or inappropriate use of resources, unnecessary quarantine of contacts (i.e., work days lost) as well as the psychological effects of being stigmatized for having SARS.

From your calculations, you find that the PPV is low for blood, throat and NP swabs. If a PCR test is positive, there is a 45-83% chance that it is a false positive. The NPV is high (i.e., under the assumption that proper sampling and techniques were used, a negative PCR test indicates that the individual was likely not infected with SARS CoV).

Answer 9c

Positive PCR in those that did not meet case definition

Among individuals that did not meet the case definition for SARS, some have positive PCR test results for SARS CoV (using NP and throat swabs). Based on the low PPV of these PCR tests, the microbiologist warns that these may be false positive results. However, since SARS is a new disease and the full spectrum of illness is not known, it is possible that the case definition is too specific and excludes those at the mild end of the clinical spectrum. That is, some of these people who did not meet the case definition may be still infected with SARS CoV.

Answer 9d

The PPV of a test is dependent on the sensitivity and specificity of the test as well as the prevalence of the disease in the population. When the prevalence of disease is low, the PPV will also be low, even using a test with high sensitivity and specificity. Therefore, if there were a case of SARS reported in a setting where there were no known cases, one should consider the possibility that this may be a false positive result. It is important to use a testing strategy which starts to test patients with a higher likelihood of being a case first, together with stepwise laboratory testing (i.e., a more sensitive test first with a second test for confirmation).

Answer 9e

The negative predictive value of the PCR tests was generally high indicating that a negative test likely means that the individual was not infected with SARS CoV. However, it is possible that an infected individual could test negative on PCR due to poor timing of specimen collection or improper handling of the specimen.

Answer 9r

Human metapneumovirus

The discovery of metapneumovirus in some samples could mean several things. The specimen could have been contaminated during sampling or in the laboratory, at some point in the diagnostic process. Another possibility is that there has been a misdiagnosis and the SARS patient in fact has metapneumoviral infection and is not a SARS case at all. However, as specimens from multiple SARS cases are positive for both organisms, it is most likely that it is a co-infection. The potential role of human metapneumovirus in SARS is not completely understood.

Antibodies to SARS-associated CoV were detected in 96% of the Toronto SARS cases’ specimens providing further strength to the body of global evidence that this outbreak was caused by a new coronavirus. Laboratory test results for SARS-CoV must always be interpreted in conjunction with clinical and epidemiological history.

Part 10 Sequence data (Optional)

RT-PCR sequencing analysis

The very first whole virus genomic sequence on the SARS CoV was done to confirm the novelty of the virus and to develop molecular-based tests, such as the PCR.

Sequence analysis of different SARS strains were conducted to see if strains found in different geographic areas of the world were genetically related. This was done for three reasons:

1. To determine the stability of the genome

2. To determine whether variability in the genome produces strains with different pathogenicity

3. To determine if strains were related and if so, whether this could facilitate the understanding or identification of transmission chains

The phylogenetic tree of the sequencing results is presented in the figure below. [pic]

Recall: To interpret the above, compare the base pair differences of each strain to the sequence results of the index case (shown in the far left column).

Question 10a

How would you interpret this and the following phylogenetic tree given what you know of the origin of the Toronto cases and the common source at the Metropole hotel?

What factors can influence variations of the genome?

Figure

[pic]

YiJun Ruan, Chia Lin Wei, Ling Ai Ee, et al, Comparative full-length genome sequence analysis of 14 SARS coronovirus isolates and common mutations associated with putative origins of infection, Lancet, May 2003 Vol 361, 1779-1785

Discussion for Part 10

Interpreting phylogenetic trees

A phylogenetic tree is a graphical way to depict the evolutionary relationships of a group of organisms, in this case, viral strains. The distance between the branches of the tree is directly proportional to the genetic distance between the strains. If two species have a small distance between them, then they have a recent common ancestor; if they are far apart, then their common ancestor is in the remote past.

The figure above shows that the strains from Singapore are more closely related to each other than the ones from earlier infections in China. It also shows that the Toronto strain is more closely related to the Hong Kong strain, which fits in well given the known transmission history. Similarly, in the figure showing the character sequences, there are fewer differences (shown in red) in base pairs of strains linked to Hotel M compared to those that are not linked to Hotel M.

Factors influencing variations in the genome:

• Viruses may mutate within the host, or after passing from person to person. These mutations can happen spontaneously or through adaptations to environmental conditions.

• Viral mutations can also occur during laboratory propagation for isolation, especially when passing through several life cycles on the cell/lines.

• Human error can occur in transcribing genomes to databanks, especially if many different investigators are involved in the complete sequencing.

Part 11 Drawing conclusions

Looking at the broader global context, surveillance data indicated that by the end of the outbreak, there were 8,098 cases and 774 deaths worldwide, with an overall case fatality ratio of 9.6% (although this varied from country to country). Mortality was highest in those aged 24 years and older. It is reported that up to 80% of infections were acquired in hospitals.

Back to Toronto, where you now have all currently available laboratory data.

Question 11a

Integrating the laboratory and epidemiological data, provide a brief summary of what you know about the outbreak in Toronto.

Question 11b

What else would you like to know about this new pathogen? What type of epidemiological studies would you suggest to address these questions?

Discussion for Part 11

Outbreak Summary

An outbreak of severe acute respiratory illness originated in Guangdong province, China in November 2002. As a result of international travel, the illness was introduced in Toronto in February 2003l. The WHO subsequently declared an outbreak of a new illness, Severe Acute Respiratory Syndrome (SARS) on March 12th, 2003.

Epidemiology – person, place, time

By May 2003, when the first phase of the outbreak was declared over, 257 SARS cases had been reported in Toronto, as a result of unrecognized spread to hospital staff, patients and visitors to Hospital A and to contacts of these cases. Approximately 63% of the infections were health-care acquired. The case fatality rate was 10.5%.

Laboratory

Almost all cases with available laboratory data had positive serology results for SARS CoV. Based on strong epidemiology-laboratory collaboration worldwide, transmission from Asia to Canada was confirmed through sequencing and comparison of the strains isolated in Asia and Toronto. PCR sequencing analysis confirmed that the outbreak was caused by a newly recognized coronavirus.

As SARS was caused by an unknown pathogen, there was added urgency to develop reliable, accurate laboratory tests in light of the severity of illness and global panic. This necessitated close collaboration of epidemiologists and laboratory specialists.

Additional information

Although the outbreak in Toronto was controlled and initially declared over in early May, 2003, a new cluster of cases was reported in a Toronto hospital on May 22nd. On July 5th, 2003, WHO reported that the chain of transmission of SARS was broken.

Further investigations that could be recommended include:

• A case-control study to look at risk factors for infection (e.g., use of personal protective measures, exposure through aerosol-generating procedures etc.) to better understand disease transmission.

• Seroprevalence studies to look at extent of transmission and explore the spectrum of illness (including the possibility of asymptomatic cases)

Epilogue – A global picture

Worldwide, 5,865 cases of SARS were reported as of May 1st, 2003.

On May 23rd, 2003, coronaviruses linked to SARS were found by researchers in China in some animals: civets, racoon-dogs, and Chinese ferret badgers. No new cases appeared once the last patients recovered. However, SARS CoV is still available in many laboratories worldwide. Laboratory-acquired infections caused by inappropriate handling continued to be sources of three laboratory-acquired cases of SARS since the end of the outbreak. It is important that adequate containment practices and procedures are followed when working with infectious agents to minimize these risks.

The rapid spread of SARS to 29 different countries in a short period of time demonstrates how a pandemic might behave. However, during SARS, epidemiologists and laboratories worked closely in conjunction with international health organizations and governmental agencies to coordinate outbreak control efforts. As a result of active case finding, epidemiological and laboratory information and international travel restrictions, a feared uncontrolled worldwide epidemic never materialized. Expertise was pooled and information was shared to discover this new coronavirus. The importance of this multisectoral collaboration remains among the top lessons learned.

References:

1. SARS Reference 10/2003, editors Kamps and Hoffman, 3rd Edition, Flying Publisher, 2003. ()

2. WHO. Severe acute respiratory syndrome (SARS): Status of the outbreak and lessons for the immediate future. Geneva, 20 May 2003.

3. Reference: WHO. Case Definitions for Surveillance of Severe Acute Respiratory Syndrome (SARS). Accessed 22nd February 2005. Last updated 1st May 2003.

4. Medical News Today. Consequences of SARS Revealed.

5. Reference:

6. WHO. Severe Acute Respiratory Syndrome (SARS)-multi-country outbreak - Update 49.7 May 2003.

7. WHO. Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003

8. Reference:

9. Reference:

10. WHO biosafety guidelines for handling SARS clinical specimens and materials derived from laboratory investigations of SARS

11. Guidance on regulations for the Transport of Infectious Substances 2007-2008



Appendix 1: Differential diagnosis for acute respiratory infections

|UPPER RESPIRATORY TRACT INFECTIONS |

|Acute pharyngitis |Viral: |

| |Influenza A & B Adenovirus Parainfluenza virus Coxsackie A |

| |Rhinovirus Coronavirus Epstein-Barr Virus |

| | |

| |Bacterial: |

| |Streptococcus pyogenes (Group A beta haemolytic streptococcus), Corynebacterium diphtheria |

|Rhinitis |Rhinovirus Coronavirus Adenovirus Respiratory syncytial virus (RSV) Parainfluenza virus |

|Laryngitis |Primarily viral: Influenza, rhinovirus, adenovirus |

|Laryngotracheobronchitis |Primarily viral: Parainfluenza virus, influenza, RSV, adenovirus, rhinovirus |

| |Bacterial: Mycoplasma pneumoniae |

|ACUTE LOWER RESPIRATORY TRACT INFECTIONS |

|Acute bronchitis |Viral: Parainfluenza virus, influenza, RSV, adenovirus, measles |

| | |

| |Bacterial: Bordetella pertussis, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydia pneumoniae |

|Influenza |Influenza virus |

|PNEUMONIA |

|Community-acquired pneumonia |60 years old: Respiratory viruses |

| |Bacterial: Pneumococcus spp., Haemophilus influenzae, aerobic gram negative rods, Staphylococcus aureus |

|Nosocomial pneumonia |Enterobacter spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp., S. aureus, Anaerobic bacteria  |

|Atypical |Mycoplasma pneumoniae, Respiratory viruses including SARS coronavirus, Influenza, Chlamydia pneumoniae |

|Other |Opportunistic pneumonias (i.e., fungal) wouldn't present as an outbreak |

Note: Also consider 1) non-infectious possibilities including new onset asthma (e.g., due to chemical exposure), thiamine deficiency in infants; 2) chemical/bioterrorism agents or 3) the possibility of a new pathogen. The list above is only a short summary of the most probable causes; it is not meant to be an exhaustive list.

Appendix 2: Specimen Collection and handling during outbreaks of respiratory illness

| |Timing of specimen |Organisms |Test |Transport medium/ |Instructions |Time to test results|Other comments |

| |collection | | |container type |for Transport | | |

|Nasopharyngeal or |As above | | |Viral: | | |While throat swabs are easy to |

|oropharyngeal swabs* | | | |Sterile vials with | | |collect for bacterial culture, |

| | | | |viral transport | | |as they are also contaminated |

| | | | |media | | |with normal flora, do not keep |

| | | | | | | |at room temp for >1hr. Process |

| | | | |Bacterial: | | |as soon as possible. If not |

| | | | |Tellurite in tube | | |possible, then keep at +4°C. |

| | | | |(except B. | | | |

| | | | |pertussis: | | | |

| | | | |Reagan-Lowe media in| | | |

| | | | |tube) | | | |

| | | | | | | | |

| |Timing of specimen |Organisms |Test |Transport medium/ |Instructions |Time to test results|Other comments |

| |collection | | |container type |for Transport | | |

|BAL |When clinically |M. tuberculosis |Viral: |Sterile leakproof | | |For testing |

| |appropriate: intubated,|S. pneumoniae |Culture |container | | |in-patients; |

| |severe lung disease, or|H. influenzae |PCR | | | | |

| |pleural effusion. |M. pneumoniae | | | | |Consider these specimen types |

| | |L. pneumophila |Bacterial : | | | |if sputum not available |

| |Isolates for viral |C. pneumoniae |Standard cultures | | | | |

| |testing should be |S. aureus | | | | | |

| |collected within 4 days|Fungi | | | | | |

| |after illness onset as | | | | | | |

| |virus shedding | | | | | | |

| |decreases rapidly after| | | | | | |

| |this. | | | | | | |

| | | | | | | | |

| |Bacteria: preferably in| | | | | | |

| |the acute phase of | | | | | | |

| |illness | | | | | | |

|Tracheal aspirate | | | | | | | |

|Pleural fluid tap | |S. pneumoniae | | | | | |

| | |S. aureus | | | | | |

| | |H. influenzae | | | | | |

| | |Anaerobic bacteria | | | | | |

| |Timing of specimen |Organisms |Test |Transport medium/ |Instructions |Processing Time |Other |

| |collection | | |container type |for Transport | | |

|Whole Blood |At fever peak |Bacteria |Standard hemoculture |Standard blood culture|Room temperature within 2|Days |Sample in aseptic conditions. |

| |preferably. | | |bottle) |hours if around 25-35°C. | |Do not cool or freeze the bottles |

| |Repeat sample | | | |If room temperature is | | |

| |(collect 2 bottles 3 | | | |higher and lower, | |Note: Blood culture is positive for |

| |times or 3 bottles 2 | | | |transport in an incubator| |the causative organism in up to 20% |

| |times, at fever peaks| | | |(37°C) | |cases of pneumonia |

| |or regular intervals | | | | | | |

| |(few hours apart) | | | | | | |

| | | | | | | | |

|Whole Blood |Any time in illness |Bacteria, virus, parasite|PCR |EDTA (lavender top |ship cold packs (domestic|Hours or few days |Do not centrifugate and do not freeze|

| | | | | |or international) | |EDTA whole blood |

| |Timing of specimen |Organisms |Test |Transport medium/ |Instructions |Processing Time |Other |

| |collection | | |container type |for Transport | | |

|Tissue, major organs | | |Culture |Culture or PCR: |Ship at room temp. | |For patients who are deceased |

| | | |PCR |Sterile container with| | | |

| | | |Histopathology |physiologic water | | | |

| | | | |Histopathology: | | | |

| | | | |Formalin fixed or | | | |

| | | | |paraffin embedded. | | | |

|Stool |Any time: |Adenovirus |Culture |Sterile jar |Ship on ice | |Isolation of adenovirus in the stool |

| |Best sample at around| |PCR | | | |is more informative if simultaneously|

| |day 14 | | | | | |isolated from the respiratory tract |

| | | | | | | | |

|Urine |Acute phase of | |Antigen detection for S. |Clean catch, sterile |Ship on cold packs | | |

| |illness | |pneumoniae and Legionella |leak-proof container |(domestic) | | |

| | | |pneumophila | |Ship on dry ice | | |

| | | | | |(international) | | |

*Swab: use only sterile Dacron or rayon swab with plastic shaft (wooden sticks should not be used as it inhibits viral growth)

**Stability of Specimen – At ambient temperature: 2 hours; Refrigerated: 3 days; Frozen: Unacceptable

In general do not store specimen for bacterial culture for more than 24 hours. Viruses, however, usually remain stable for 2-3 days at 40C

Appendix 3: Taking a nasopharyngeal (NP) swab

1. Put on all protective equipment.

2. The specimen should be collected with a nasopharyngeal swab. Only sterile Dacron or rayon swabs with plastic shafts should be used. Wooden sticks should not be used as it inhibits viral growth). Swabs intended for bacterial culture should not be used.

3. Incline the person’s head and insert the NP swab through the nostrils until resistance is met by virtue of contact with the nasopharynx. (*Note: sometimes you may hit nasal turbinates first, and need to change angle a bit to get past them). Quickly rotate the swab 3-5 times against the nasopharynx as long as tolerated by the patient. Note that coughing or patient resistance is adequate incentive to remove the swab.

4. Withdraw the swab and insert into the transport medium (viral transport media, or Regan Lowe media for B. pertussis) by inserting the swab at least ½ inch below the surface of the medium. Bend or cut off the wire to fit the transport medium tube and reattach the cap securely.

5. Label the transport tube with the name and specimen source as per requirements.

6. Complete the specimen submission form with all required information. Failure to submit or complete this form could result in a delay in processing the specimen.

Appendix 4: Biosafety levels and Risk groups (From the WHO Laboratory Biosafety Manual, 3rd edition, 2005)

| |BIOSAFETY LEVEL |

| |1 |2 |3 |4 |

|Isolation of laboratory |No |No |Yes |Yes |

|Room sealable for |No |No |Yes |Yes |

|Decontamination | | | | |

|Ventilation : | | | | |

|— inward airflow |No |Desirable |Yes |Yes |

|— controlled ventilating system |No |Desirable |Yes |Yes |

|— HEPA-filtered air exhaust |No |No |Yes/No |Yes |

|Double-door entry |No |No |Yes |Yes |

|Airlock |No |No |No |Yes |

|Airlock with shower |No |No |No |Yes |

|Anteroom |No |No |Yes |_ |

|Anteroom with shower |No |No |Yes/No |No |

|Effluent treatment |No |No |Yes/No |Yes |

|Autoclave: | | | | |

|— on site |No |Desirable |Yes |Yes |

|— in laboratory room |No |No |Desirable |Yes |

|— double-ended |No |No |Desirable |Yes |

|Biological safety cabinets |No |Desirable |Yes |Yes |

|Personnel safety monitoring capability |No |No |Desirable |Yes |

|RISK |BIOSAFETY LEVEL |LABORATORY TYPE |LABORATORY PRACTICES |SAFETY EQUIPMENT |

|GROUP | | | | |

|1 |1= Basic |Basic teaching, research |GMT |None; open bench work |

|2 |2 = Basic |Primary health |GMT plus |Open bench plus BSC for potential aerosols |

| | |services; |protective clothing, | |

| | |diagnostic |biohazard sign | |

| | |services, research | | |

|3 |3= Containment |Special diagnostic |As Level 2 plus |BSC and/or other primary devices for all |

| | |services, research |special clothing, |activities |

| | | |controlled access, | |

| | | |directional airflow | |

|4 |4 = Maximum |Dangerous |As Level 3 plus |Class III BSC, or positive pressure suits in |

| |containment |pathogen units |airlock entry, |conjunction with Class II BSCs double ended |

| | | |shower exit, |autoclave |

| | | |special waste |(through the wall), filtered air |

BSC= Biological Safety Cabinet

GMT= Good Microbiological Techniques

-----------------------

Sensitivity=a/(a+c)

Specificity=d/(b+d)

PPV=a/(a+b)

NPV=d/(c+d)

The letters before the numbers refer to the place where the strains were isolated:

SIN= Singapore

BJ=Beijing

GZ= Guangzhou

CUHK= Chinese University of Hong Kong

HKU= Hong Kong University. TOR= Toronto

You will need the following:

Mask, goggles, gown, gloves

nasopharyngeal swab

viral transport medium (or Regan Lowe media for B. pertussis)

Linked to Hotel M

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