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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.



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.

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.

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 (TD) or 'melt', and the range over which the oligonucleotide primer can hybridize (TM) 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 (TA), 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 (TE), 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.

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 TM (melting temperature) but is in fact 5�10�C below the TM. The term TM 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.

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.

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