Part 1: Exploring Healthcare Delivery at Indian Health Service, Navajo Nation (Gallup, NM)

My first week at Indian Health Service (IHS) in Gallup, New Mexico was incredibly diverse. I spent the first couple days in clinic, where I learned about the detection and management of the most common diseases in the community--namely, diabetes, obesity, rheumatologic illnesses, alcohol dependence, and depression. Unfortunately, nearly all the patients I met had an underlying diagnosis of diabetes and obesity, likely due to a combination of genetics, an increasingly sedentary lifestyle, and lack of access to healthy food (more on this in my next blog post). I also learned how the PCP diagnoses and treats rheumatoid arthritis (including how to differentiate between general non-active RA and a flare) and ankylosing spondylitis. Since there is no local rheumatology specialist, the PCP is responsible for managing this on his/her own. The PCP I worked with also taught me about the use of Naltrexone in alcohol dependence, which I had previously never prescribed during my residency training in Boston. I also met several patients who were successfully treated with Naltrexone and were doing a lot better. Finally, regarding depression, there were a number of patients I met who had undergone physical and/or verbal abuse from their family members as children and as adults, usually in the context of alcohol abuse. I learned about the stigma of getting therapy in the community, which made me realize how important and needed it is to integrate behavioral health with primary care.  

I spent the second half of the week doing a mixture of inpatient medicine and ID/tuberculosis clinic. On the inpatient side, I took care of a young man who was admitted overnight for fever, headache, jerking movements and diarrhea. He had a history of coccidio-meningitis and resultant syringomyelia status post VP shunt and so meningitis was on the differential and he was initially treated for both bacterial and recurrent coccidio-meningitis. Serotonin syndrome was also on the differential since he was on a number of pain medications for his neuropathic pain (including a high dose SSRI and SNRI), so we discontinued these potentially exacerbating medications. Ultimately however, his CSF studies from the lumbar puncture were benign and his stool study came back as c.diff positive so he was treated for c.diff and sent home. I am glad that ultimately the patient had an identifiable and treatable source of his fevers and it was fascinating to think through his case as it progressed in the hospital. It was fun to present his case during ICU and infectious disease rounds at Gallup Indian Medical Center and get the input from the team of physicians and other healthcare providers.

In tuberculosis clinic I learned the nuances of how to treat latent TB infection as well as active TB and how to monitor for drug side effects. I took care of patients who had been recently hospitalized for extrapulmonary TB and were undergoing treatment, and I also initiated treatment in a patient who is a healthcare worker who had a positive ppd. It was interesting to learn about how common TB is in the population and about the process of contact investigation once someone is diagnosed. We also learned about TB reactivation in immunocompromised patients, namely in rheumatoid arthritis and diabetes, which are very common on the reservation. Finally, we learned about the history of Hantavirus, which was first recognized by Bruce Tempest in 1993 at Gallup Indian Medical Center.

Read More

Indian Health Service and Navajo Nation

5/9/15

It is a Saturday morning in early May here in Gallup, NM, and as I sit down to write this post, I am staring at a flurry of snow outside my window and a blanket of white upon the town. In Boston, the snow often cloaks and transforms the city, but here in Gallup, the snow only shades the imperturbable land and sky. Indeed, what struck me most when I came here was the vastness of the land, of the sky, of the history. It is overwhelming and humbling at the same time. 

Gallup is located squarely in the Navajo Nation, a tribal sovereign nation of close to 200,000 people who identify themselves as Navajo. It is bordered by other culturally and linguistically distinct tribes, the Zuni and the Hopi. 

Much like the land, the people here are enduring and have weathered storms in the past - from rival tribes, to colonization by European powers, to subjugation at the hands of the United States, to more modern threats including pollution from coal and uranium mining and now substance abuse, HIV/AIDS, and diabetes. 

Through it all, Dine, or "the people� in the Navajo language have persevered.

There have been bright spots in Navajo history, from the heroic role of code breakers in WWII to the establishment of the sovereign nation of Navajo to the retention and active use of Navajo language and traditions in everyday life.

The Indian Health Service and its hub in Gallup has pioneered a number of incredible innovations along the way to better service its constituents including the use of community health workers, the active treatment of HIV/AIDS, Hepatitis C, and Tuberculosis, and the implementation of telemedicine to enhance care.

Over the next few blog posts, I hope to explore Navajo history and culture and highlight my clinical experiences.

Stay tuned.

Read More

Snapdate: Confirmed Ebola virus disease cases - the end in sight?

I think we're a little bit beyond "jinxing" something by pointing it out, so here is graph of the confirmed Ebola virus disease cases based on the World Health Organization report date (Situation summary or Situation Report), including a basic model to predict when cases may hit zero, if nothing changes.

The P-value for this linear trend model is 0.00067. The standard error = 19.29;R-square = 0.14.
Click on graph to enlarge

I use Tableau Desktop Public Edition v9.0.0 for my graphs these days - and have taken advantage of its inbuilt linear trend model for this chart. This "model" accounts for precisely nothing apart from the trend based on the numbers that are available and have been plotted in this particular way, on this day, near a full moon. 

Reported numbers or outbreaks could flare up tomorrow or dry up overnight. 

I can say that over the past 2 weeks, data from each new summary or report have moved the predicted "end" data closer - from mid-June to now early June.

I am not an expert at modelling or statistics so please just take this at face value. The line suggests that if all things stay the same, we will reach zero considered cases per report around the 3rd of June 2015.

Please let it be so. 

Realistically, we may be heading for another "step down" - followed by a smaller trickle of ongoing cases for some period, ahead of a final push to zero. But there are experts who will know more about this than I.

Once we get to zero, the 42 day count begins.

Read More

The mechanics of the polymerase chain reaction (PCR)...a primer

The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold. As the name suggests, the process creates a chain of many pieces, in this case the pieces are nucleotides and the chain is a strand of DNA.

PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must occur at the enzyme's ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (DDDP) derived from thermophilic (heat-loving) bacteria. As such, the enzymes function at higher temperatures than the enzymes we commonly use in the laboratory or have working in our bodies. These DNA polymerases operate at 60-75�C, and can even survive at temperatures above 90�C. This is important because a part of the PCR requires that the reaction reaches ~95�C as we shall see.

Apart from the DNA polymerase, PCR needs a DNA template to copy, and a pair of short DNA sequences called oligonucleotides or "primers" (described here) to get the DNA polymerase started.

Broadly speaking, there are 3 steps identified by incubating at different temperatures. The 3 steps make up a PCR "cycle".

1. Double-stranded DNA separation or denaturation (D in Figure 1)
2. Primer annealing to template DNA (A in Figure 1)
3. Primer extension (E in Figure 1)

Figure 1.A PCR cycle.

The three temperatures which make up a single cycle. The DNA denaturation section (D), oligonucleotide annealing section (A) and the primer extension (E) section are marked. The temperature range over which dsDNA duplexes can denature (T_D) or 'melt', and the range over which the oligonucleotide primer can hybridize (T_M) are also marked.

Denaturation..

At temperatures above 90�C, double-stranded DNA denatures or "melts". That means the weak hydrogen bonds that usually hold the two complementary strands together at normal temperatures are disrupted resulting in two single stranded DNA strands (shown below in an idealised form).

Primer Annealing..

At the annealing temperature (T_A), primers that collide with their complementary sequence can hybrdise or "bind" to it. The chance of such an encounter happening is increased because we use a vast excess of each primer in the reaction mixture compared to the number of template molecules present.

The assay in the example below has been designed to amplify a region of the template spanned by and including, the primer sequences.

Primer Extension..

At the extension temperature (T_E), the DNA polymerase binds to the hybridized primer and begins to add complementary nucleotides (i.e. every time the polymerase reads a "G" on the template strand, its adds a "C"; an "A" for a "T"; a "G" for a "C" and a "T" for an"A"), chemically binding each new addition to the last to form a growing chain. The process only occurs in one direction. In our example, the green primer is binding to its complementary template sequence and is facing toward the right (this is called the 5' (five-prime) to 3' (three prime) direction. Extension occurs in the direction that the primer faces. The result is a new double-stranded PCR product we usually call an "amplicon". An amplicon can be defined as an amplified molecule of a single type, in this case, an exact replicate of the original template.

Exponential Template Duplication..

The process is then repeated by cycling through the temperatures over and over again (35 to 55 times). Each cycle results in a new DNA duplex, each strand acting as a potential template for one or other primer.

Some interesting things stand out from the figure below.

The original template strands (blue and red) continue to act as templates because the PCR process is not destructive. However, each cycle produces a greater number of the shorter amplicon molecules. These are shorter in our example because the primers shown, bind within the template sequence. Eventually the majority of the amplicon in the reaction vessel will be the expected length, i.e. just the region spanned by, and including the primer sequences.

It is possible to mathematically predict the pattern of amplicon accumulation. In our example, we have started with two strands. In a perfect PCR reaction (which rarely occurs!), we have two new strands making a total of four. After the second cycle we have eight strands, then 16, 32 and so on. The reaction is doubling the number of strands each cycle or to make that an equation, we have 2n

Note: to make the process easier to understand, I have drawn the DNA strands as straight lines - in reality, DNA does not exist in as simple a form as this.

Further reading...

1. PCR primers...a primer!
   http://newsmedicalnet.blogspot.com.au/2015/05/pcr-primersa-primer.html
2. Reverse transcription polymerase chain reaction (RT-PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/reverse-transcription-polymerase-chain.html
3. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
4. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
5. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts� roundtable: Real-time PCR and microbiology�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
6. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. �Challenges facing real-time PCR characterisation of acute respiratory tract infections�, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
7. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: �Real-time PCR; History and fluorogenic chemistries�, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
8. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:�Quantification of microorganisms: not human, not simple, not quick�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
9. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
10. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.

Read More

PCR primers...a primer!

A DNA Down Under post

PCR (described here) functions mainly because of two components - a thermostable DNA polymerase and a pair of DNA 'primers'. Primers are short, made to order, stretches of oligonucleotides ('oligos' - from Greek meaning scanty or few). Modern oligos can be synthesized in lengths >100nt however the behaviour of oligonucleotides longer than 20nt is different from that of shorter oligos and different calculations are employed to determine their thermodynamic characteristics.

What is a primer...?

Primers, as their name may suggests, prime the nucleic acid template for the attachment of the polymerase. This is the first step towards duplicating that template. The primer directs the polymerase to move in a 5' to 3' direction (drawn left-to-right; Figure 1) because of the 'direction' of

DNA (See DNA Structure for more background).

Figure 1. DNA has direction. The polynucleotide chain shown 
above is 'read' in a 5' to 3' direction by the polymerase. This would 
be from the top to the bottom or from the phosphate group 
to the hydroxyl group.

Primer binding...

Primers hybridize at a temperature that is affected by their sequence, concentration, length and ionic environment. This annealing temperature is usually referred to as the T_M (melting temperature) but is in fact 5�10�C below the T_M. The term T_M describes the temperature at which 50% of the primer�target duplexes have formed.

Primer specificity...

PCR gleans its extreme specificity from the primers. At each and every position of a new primer, we have 4 nucleotides to choose from, dATP, dCTP, dGTP and dTTP. 

Figure 2. Deoxynucleotide triphosphates. Each of the five deoxynucleotides are shown.2'-deoxycytidine-5'-triphosphate (dCTP; C9H16N3O13P3, MW=467),2'-deoxyguanosine-5'-triphosphate (dGTP; C10H16N5O13P3, MW=507),2'-deoxyadenosine-5'-triphosphate (dATP; C10H16N5O12P3, MW=491),2'-deoxythymidine-5'-triphosphate (dTTP; C10H17N2O14P3, MW=482) and 2'-deoxyuridine-5'-triphosphate (dUTP; C9H15N2O14P3, MW=468). 

So, if we design a sequence-specific primer of 20-30nt nucleotides in length ('20-30mer'), the chance that that exact sequence will occur randomly in nature will be 1/4 x 1/4 x 1/4 etc, 20 or 30 times i.e.

That means a 1 in 10¹² to 10¹⁸ chance of a 100% homologous match to an unintended target. Or to put that in perspective, there are 2.85 x 10⁹ basepaired nucleotides in the entire human genome.

While that all sounds very convincing, in reality, primers designed to detect viruses often share significant amounts of homology with the human genome - sometimes resulting in false positive amplifications. Even when the homology is far from 100%, primers may still amplify an unintended target as shown below. This most likely reflects the co-evolution of many viruses with humans during which time they have "captured" bits of our genome and "deposited" bits of their own genome. 

Next we'll list a few of the problems we can encounter when using the PCR.

Primer dimer...

The first problem I'll discuss is the most common and the most difficult to avoid. Depending on your requirements, it may also be the least significant.

When a small amplicon results from the extension of self-annealed primers, you get primer-dimer (PD) i.e. a dimer of one (self-annealing) or both primers resulting in a template capable of being extended by the polymerase. PD formation is highly efficient because the primers are in vast excess compared to the amount of template or even to the number of amplicon molecules at the end of the PCR.

This excess drives the formation of PD. Two main concerns arise from PD formation.

1. Because PD formation is so efficient, it rapidly consumes dNTPs and primers and generates amplification inhibiting pyrophosphates. All of which can prematurely plateau the exponential accumulation of product.
2. If we using a dsDNA-associating fluorescent molecule to follow the PCR's progress during real-time PCR, then PD will also show up, and, at least during the kinetic portion of the assay, cannot be differentiated from the signal of specific amplicon accumulation.

Figure 3. Examples of how primer-dimer (PD) amplicon can be formed.
Ten examples are shown of sense and antisense primer interactions
resulting in an amplicon. Note that different length amplicons can be formed.
The largest PD would result from the hybridization of the smallest number of
nucleotides and would approach the length of the two primers added together.

Mispriming...

Mispriming is the result of a primer binding to an unintended template resulting in amplification. The amplicon (PCR product of a single species) can sometimes be the same size as the intended product, but is usually a different size when viewed following agarose gel electrophoresis.

Mispriming occurs because of poorly optimised conditions or because we haven't checked whether our sequence will inadvertently bind to an entirely different target entity e.g. a region of the human genome instead of the intended virus genome. Sometimes it just happens.

Mispriming can usually be avoided by more intensive comparison of the primer's sequence against the GenBank database using the Basic Local Alignment Search Tool (BLAST) at NCBI. Of course, a BLAST comparison will only find matches among those sequences housed in the database. When it comes to PCR where a single nucleotide mismatch can cause an amplification to fail, or at least perform with reduced efficiency, BLAST'ing primers can lead to a feeling of very false security.

In some instances the homology of the primer to its template may indicate a perfect match simply because viral variants have not yet been sequenced and submitted. Also, because there may be many undiscovered viruses and unsequenced non-viral genomes in the world, obviously none of which are represented on GenBank, a specific match, or a "no match", does not mean that you have exhausted your search for homologues. Take it all with a grain of salt. Designing two pairs of primers around the target region is a good place to start. This helps address the unexpected.

Structural problems...

This problem results from the way we design our primers. I am excluding self-annealing and secondary structures from here because we will deal with them specifically in the next section. --work in progress---

Further reading...

1. A crowd-sourced database of virus primers. www.virusprimers.org/
2. The mechanics of the polymerase chain reaction (PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
3. Reverse transcription polymerase chain reaction (RT-PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/reverse-transcription-polymerase-chain.html
4. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
5. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
6. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts� roundtable: Real-time PCR and microbiology�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
7. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. �Challenges facing real-time PCR characterisation of acute respiratory tract infections�, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
8. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: �Real-time PCR; History and fluorogenic chemistries�, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
9. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:�Quantification of microorganisms: not human, not simple, not quick�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
10. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
11. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.

Read More

Reverse transcription polymerase chain reaction (RT-PCR)...a primer

A DNA Down Under post

The polymerase chain reaction (PCR) is a technique for copying a chain of DNA as many as a billion times. It purpose is so that we can use some form of technology to detect what would otherwise be too little material to see in the first place. The process of PCR is covered on the PCR page so I won't repeat it all here.

Before PCR for virus detection from human samples, we usually prepare the nucleic acids with an extraction or purification step. The shorter this is the better when testing, or screening, a lot of samples.

PCR can be used to detect some viruses straight out of the box. In these cases, the viruses need to have genes or a genome that is made of DNA. But many viruses don't store their genetic code as DNA, they use RNA as the plan from which they make more of themselves and their viral proteins. In these cases, DNA only plays an intermediate role, if any.

DNA viruses include the adenoviruses, herpesviruses, HIV (an example which also has RNA stages), polyomaviruses, bocaviruses, parvoviruses, papillomaviruses, poxviruses, megaviruses and many others. PCR also works well for plasmids and human genes and other DNA fragments we want to work with in the lab for reasons other than a diagnosis where we ask if the virus is in the human or not.

RNA viruses include influenza viruses, parainfluenza viruses, rhinoviruses, enteroviruses, cosavirus, klasseviruses, parechoviruses, respiratory syncytial virus, coronaviruses, human metapneumovirus and also many others. We also look at gene activity which involves detecting and measuring gene/genome transcription via reverse transcriptase (RT) PCR-based quantification, usually of messenger RNA (mRNA). But because PCR is based around the use of a heat stable DNA-dependent DNA polymerase, we would need to first make that RNA into DNA so that the main enzyme can use it and duplicate it and make enough of it to detect or use in molecular biology...or whatever the downstream application may be.

To make a DNA copy of the RNA, we need to add in another enzyme and another step to the PCR process. That enzyme, the RT is used in a step called reverse transcription.

The addition of this step also changes the initialism of the technique to RT-PCR. This is not to be confused with real-time PCR which is shortened to rtPCR. So an RT-rtPCR, which we use when detecting or quantifying RNA viruses in human samples, is a reverse transcription real-time polymerase chain reaction.

A standard PCR then has added to it a 10-30min incubation at an appropriate temperature (40-50'C), a denaturation step to kill of the enzyme (and sometimes to active the DNA polymerase; 92-95'C for 2-15min) and separate any DNA that is double stranded, followed by the multi-cycle PCR amplification process which can use the new DNA strand as a template for exponential copying....the billion-fold amplification reaction.

Further reading..

1. PCR primers...a primer!
   http://newsmedicalnet.blogspot.com.au/2015/05/pcr-primersa-primer.html
2. The mechanics of the polymerase chain reaction (PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
3. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
4. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
5. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts� roundtable: Real-time PCR and microbiology�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
6. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. �Challenges facing real-time PCR characterisation of acute respiratory tract infections�, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
7. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: �Real-time PCR; History and fluorogenic chemistries�, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
8. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:�Quantification of microorganisms: not human, not simple, not quick�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
9. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
10. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.

Read More

The third outbreak of influenza A(H7N9) virus seems to be over...

Cumulative curves of reported H7N9 cases and deaths in humans.
Click on graph to enlarge.

By the looks of the curve on the right, the rush of cases that defined the third known outbreak of the low pathogenicity avian influenza A virus subtype, H7N9, is over...for another season anyway. 

If we get into the nitty gritty, as I have below, there are a couple of interesting things to see. First though - let us remember that these are just reported data:

• there may have been some cases that were not reported for whatever political, medical, social or personal reasons - these data are an idea of what happened - look at the rends and don't get hung up on the specific values
• the overwhelming majority of the cases have reported to a healthcare facility with respiratory disease due (presumably) to either infection - we have no idea how many other people have been infected, what proportion were mild or asymptomatic (as I've discussed e.g. here and here, so we know it is possible). It could be half as many again, or 100 or 1,000 times as many.
• these are only cases that have been examined with a laboratory test (as far as we know) - there may have been many other cases of "influenza-like illness" that did not get sampled and tested but were managed under (or not) the assumption that they were influenza of some type, subtype or strain.

Please note-the graphs used here can all be found on my fixed interactive H7N9 page at:http://newsmedicalnet.blogspot.com.au/2014/11/influenza-ah7n9-virus-detection-numbers.html

The interesting stuff includes:

• For the 2 outbreaks we have continuous data for - 2013-14 and 2014-15, the start of the outbreak seems to be around October/November, with the peak around January/February. 
• Outbreak 3 did not seem to reach the heights of the preceding year however, from what we could glean from pretty poor data, the link to poultry exposure was as strong as ever. Perhaps market closures in response to deaths were a little more effective/efficient/wide-ranging in Outbreak #3? Pure speculation

Click on graphs to enlarge.

• Most of the cases in 2015 (bottom maps) were on the east coast of China

Click on map to enlarge

• Most of the activity in the 3rd outbreak was focussed in Guangdong, Fujian and Zhejiang provinces (the red ones above) 
• Xinjiang Uyghur Autonomous region (in the far west) and Guizhou and Hubei provinces joined the list of host regions in Outbreak #3
• Xinjiang joined Guangxi and Jilin provinces (which reporting cases in Outbreak #2) as regions of China that share a border with another country - heralding the movement of this H7N9 variant beyond China's borders possibly into Vietnam, North Korea, or a -stan

Click on graphs to enlarge.

Keep an eye out for H7N9 Outbreak #4 - coming to a colder China around November 2015. 

But for now, it might be time to hit the FluTrackers line lists (okay, I've had 4 tabs open for ages) and graph the course of another source of concern - H5N1 cases in humans.

Read More

Hubei province listed its first H7N9 case in April...some rare detail

A new province was recently added to the list of those reporting cases of avian influenza A(H7N9) virus infection in humans. 

Of course reporting does not mean capturing. Reporting has been weak this season. The cases that have shown seem to be just those who were ill enough to visit a Doctor/hospital and get a laboratory test. This is the same story for most infectious agents. We see just the tip of the iceberg, the beginning, the head of the arrow, we only scratch the surface, the glycoprotein on the envelope of the virus as it leaves the...okay, you get the picture.

This year has seen a very disappointing effort by China to provide useful public data that could permit tracking of what has become the annual outbreak of human cases of avian influenza A(H7N9) virus infection.

The human H7N9 case hotzone, at least since we heard about the virus infecting a human in February of 2013, have been on the east coast of China. We currently stand at 659 reported human cases, and over 200 deaths. Very. Roughly.

Click on image to enlarge.

I remember fondly a time when there were scads of data on H7N9-related human cases and deaths. Okay, China did over-share on a number of occasions....

Click on image to enlarge.
Story to be found here.

Click on image to enlarge. 
Story to be found here.

Click on image to enlarge. 
Story to be found here.

..but things have changed. 

For a comparison take the Kingdom of Saudi Arabia's Ministry of Health and their efforts to provide public data on Middle East respiratory syndrome (MERS) and its coronavirus (MERS-CoV). While there are a few gaping holes in the data set (c'mon guys-fill these in!), there can be as many typos as on this blog (but I'm not a public health Ministry - in case you were wondering) and the data can be intermittent, it represents the best public source of detailed, yet deidentified, human data on an ongoing zoonotic viral emergence. And that's saying something. But congratulations nonetheless!

I'm not including Ebola virus disease data-gathering here - the fact that we have had so much data - despite the initial lack of infrastructure and people trained to collect, collate and report that detail - is a fantastic testament to the efforts of those on the ground in Guinea, Sierra Leone and Liberia.

But this season H7N9 data that have been reported by public health sources have been released in blocks and lack any consistent or useful detail, except the province. Some detail is available when harvested from media reports by the ever assembled FluTrackers team. I rely heavily on their line list (to be found here). 

One example of the poor data quality this season, take a look at this text from a recent Disease Outbreak News provided by BigBlue (that's the World Health Organization, or WHO, for those not accustomed to my street groove)... 

No-one will be reading that and feel overly informed.

One is left to assume that this is how these data are coming out of China - infrequently and without detail. We regularly see that when better data are provided - and again, I hold up MERS-CoV case descriptions here - they get publicly listed by BigBlue. 

And before you head to your keyboard to ask "Why should we have access to these data?"...I will first ask you - why shouldn't we? They are collected and collated internally. They are of interest to epidemiologists, model builders, public health planners and data tinkerers the world over. And it's not as though the details are subsequently released in peer-reviewed publications. They are not. 

It's just disappointing.

Read More