Wednesday, September 19, 2012

The Rise of the BORG (Bacterial and Viral Resistance)

"It is always wise to look ahead, but difficult to look further than you can see." ~Sir Winston Churchill


Resistance is Futile

Of all the things that convince me about the future, nothing is so stark, so definite, so telling, and so real than the human hubris. No don’t get me wrong; we live in an environment of self-proclaimed wizards, the same snake-oilmen, quasi-intellectuals who can charm the socks off of anyone. We are the experts that hail from the top of Mount Sinai and yet in that very expertise of existence there is this gnawing, irritating human trait called humanism, or more aptly describes as arrogance, the one that is seduced by the vapors of grandeur, by the deluding sense of perfection, by the lofty sense of perfect knowledge. Oh but I digress into the jungle of human existence.

We exist on this planet with billions of other species. And to be certain there are estimates derived from fossilized records that over a billion species have perished between life and now and that three species go extinct every hour. We are one of those species too but with a short lifespan overall thus far as we hasten towards self-annihilation. True we are the ones gifted with the bipedal motion, the extensions of arms and the dexterity of hands that has culminated in populating this planet with monoliths as high as the Stone Henge, Empire State Building, Khobar Towers and the like and made cities where once water and swamp reigned. Yes we are the quintessence of dust as Shakespeare called us, not because we are great or majestic, but because we have the power of reason and that sprinkle of pixie dust called hubris.

"Look deep into nature, and then you will understand everything better." ~Albert Einstein

And then this:

We must rid mankind of all maladies!

Really?

I mean have we thought this one through at all. Have we reasoned? Have we spent an evening thinking about the entire collective life on this planet as a whole? Have we?

Tuberculosis



Take for example the TB conundrum. We started out with this scourge and found some medicines to combat it. And we were successful. A tincture of Streptomycin and the scourge was no more. Where previously sanatoriums had been constructed to isolate the infected and infirmed till their self-healing or death, now the magic of an injection arrested the continuum of that horrid vigil.



Soon better drugs reigned, like Ethambutol or EMB, Isoniazid or INH and Rifampin or RMP. Each garnered a better share of the TB bug. We all celebrated the onslaught against this vile bacterium that had cost so many lives and we had won. The flag of security had been flown atop the TB gravesite.

But then came the discovery of these clever critters finding little loopholes and infecting humans again. Mathematical Modeling revealed a stark but deafening verdict. TB was undergoing mutations and becoming resistant to our glorified, impenetrable lines of defenses. And that Mathematical Model was verified in real life!

“The rationale for using multiple drugs to treat TB is based on simple probability. The frequency of spontaneous mutations that confer resistance to an individual drug are well known: 1 in 107 for EMB, 1 in 108 for STM and INH, and 1 in 1010 for RMP.”

And not only that, but these little bacterial buggers were becoming more aggressive and thus more deadly in their behavior. What once was a relatively long survival, albeit in the sanatorium, to complete riddance of the TB bacterium now killed humans with impunity within a short span of days.

A study in Los Angeles found that about 6% of cases of MDR-TB were clustered. Multi-drug resistant tuberculosis (MDR-TB) is defined as TB that is resistant at least to INH and RMP. Isolates that are multi-resistant to any other combination of anti-TB drugs but not to INH and RMP are not classed as MDR-TB. Yet the MDR bug had climbed its way into the inner sanctums of inner city!

“There is currently an epidemic of XDR-TB South Africa. The outbreak was first reported as a cluster of 53 patients in a rural hospital in KwaZulu-Natal of whom 52 died. What was particularly worrying was that the mean survival from sputum specimen collection to death was only 16 days and that the majority of patients had never previously received treatment for tuberculosis.”

To be accurate about the Mathematical Model here is a short excerpt to understand the conundrum better:

“A patient with extensive pulmonary TB has approximately 1012 bacteria in his body, and therefore will probably be harboring approximately 105 EMB-resistant bacteria, 104 STM-resistant bacteria, 104 INH-resistant bacteria and 10² RMP-resistant bacteria. Resistance mutations appear spontaneously and independently, so the chances of him harboring a bacterium that is spontaneously resistant to both INH and RMP is 1 in 108 x 1 in 1010 = 1 in 1018, and the chances of him harboring a bacterium that is spontaneously resistant to all four drugs is 1 in 1033. This is, of course, an oversimplification, but it is a useful way of explaining combination therapy.”

So a new barrage of drugs are being crafted and drafted in the battle to circumvent this new abrogation in the paradigm of human thought; that we have control.

Hepatitis B Virus



Just so we don’t get too comfortable with the thought that this is only a single bug. Heck we can smash it in no time. Let me take you into the machinery of the HBV also known as the Hepatitis B Virus.


The gene that encodes the HBV polymerase overlaps with the gene that encodes the viral envelope, and so mutations in the overlapping reading frame can change both proteins.


The HBV is composed of 3400 base pairs and five genes. Each has a specific purpose.



The hepatitis B virus (HBV) is a DNA virus that replicates its genome via an RNA intermediate using reverse transcription. What is interesting is that for its replicative maneuvers, it converts itself into these CCCDNAs or Covalently Closed Circular DNA that is tightly wound together DNA machinery and is impervious to the drugs targeted against the HBV. 

Life Cycle within a human body


So while all viral particles floating around in the blood stream may be rid by the directed therapy, the CCCDNAs residing in the liver tissue by the billions laugh it off in comfort saying “We’ll get you in the end.” The CCCDNA survive to form a large reservoir for replication, reactivation and their own survival in the human body when the selective pressures of therapy are presented.

Hepatitis B Virus


It is important to note that the signs and symptoms from the HBV infection come not from the viral replication but from the Immune response initiated by the body to kill the virus itself. The genetic mutations occur at a glacial pace based on linear time. The rate is 1 nulceotide mutation per year (Mutation Rate = 5*10^-5).



The selection pressures allow the virus to mutate and create quasispecies to live within the liver: It is the constant destruction of the liver cells and the immune response mediated that determines the clinical behavior. “Mathematical modeling showed that the half-life of hepatocytes varies from 30 to 100 days, depending on individuals' immune response.”

The composition of the viral quasispecies evolves over time depending on the selective pressure including, vaccination and antiviral therapy and the host immune response. Escape mutants may then spread in the liver and become the dominant species depending on their fitness (i.e., their capacity to replicate and dominate wild-type strain in the presence of antiviral pressure) and the replication space available for their dissemination in the liver.

PCR Genotyping


The issues of genotyping plague a well-balanced assessment of the HBV quasispecies because of the many pitfalls in the process itself. Yet these are the known methods of determination at present and we have to make the most from them. The dilemma of study is compounded by the dilemma of the limitations of knowledge. Genotyping of the whole virus is tedious but remains the gold standard today. Yet studies continue to use partial genome analysis for reportage that hurts validity and confounds the truth in literature. Besides a single SNP or dual SNP cannot be ascertained by whole genome testing. It has to be done on a SNP by SNP basis with specific endonucleases... so there, now you have the most of it, if not all!


PCR Genotyping Methodology


“(Genotyping relies on either DNA sequencing or hybridization. Sequencing-based methods include standard population-based polymerase chain reaction (PCR), cloning of PCR products, and restriction fragment-length polymorphism analyses. Direct PCR-based DNA sequencing can detect a particular mutant only if it is present ≥20% of the total quasi species pool.61 Cloning can overcome this problem, but analysis of large numbers of clones is required. Viral mutants that constitute as little as 5% of the total population can be detected by restriction fragment-length polymorphism analyses, but separate sets of endonuclease reactions must be designed specifically for each (and known) mutant of interest. These methods are labor intensive, require highly skilled personnel, and are not suitable for high-throughput screening.)”

The virus exhibits the following drug resistant mutations referenced below in the Addendum: primary resistance mutations (rtM204I), secondary resistance mutations  (rtL180M with rtM204V) and compensatory mutations (rtV173L)

So after the facts and the implied human hubris, we are left with a rather sobering thought; species are coevolving and each species finds a niche to survive and progress. It is after all in the Darwin’s dictate that, “Survival of the Fittest.” Choosing to ignore such an important message based on aggregated evolutionary evidence is the hallmark of a callous disrespectful self-indulgent human mind. Say it ain’t so!

"All, everything that I understand, I understand only because I love." ~Leo Tolstoy

References:

David HL (November 1970). "Probability Distribution of Drug-Resistant Mutants in Unselected Populations of Mycobacterium tuberculosis". Applied Microbiology 20 (5): 810–14.

Sarah McGregor. "New TB strain could fuel South Africa AIDS toll". Reuters. Retrieved 2006-09-17

http://www.ihlpress.com/pdf%20files/hepdart09_presentations/emergence_prevention_vr/2_locarniniHepDart%20FINAL_SLocarnini_031209_REVISED.pdf

http://www.natap.org/2009/HBV/111109_01.htm

Addendum: (On HBV Drug Resistance)

The available agents currently in use against the HBV:

Interferon alfa-2b INTRON® A Merck/Schering 1991
Lamivudine EPIVIR-HBV® GlaxoSmithKline 1998
Adefovir dipivoxil HEPSERA ™ Gilead Sciences 2002
Entecavir BARACLUDE ™ Bristol-Myers Squibb 2005
Peginterferon alfa-2a PEGASYS® Genentech/Roche 2005
Telbivudine TYZEKA ™ Idenix/Novartis 2006
Tenofovir VIREAD ™ Gilead Sciences 2008

The viral mutations causing resistance over time to antiviral agents:

Interferon alfa-2a  (Intron A) Interferon 1991 None
Lamivudine  (Epivir-HBV) Nucleoside reverse
transcriptase inhibitor 1998 14 - 32% at Year 1;  60 - 70% at year 5
Adefovir (Hepsera) Nucleoside reverse
transcriptase inhibitor 2002 0% at year 1;  29% at year 5
Entecavir (Baraclude) Nucleoside reverse transcriptase inhibitor 2005
1.2% in treatment na├»ve at year 6;  57% in
lamivudine resistant at year 6
Peginterferon alfa-2a (Pegasys) Interferon 2005 None
Telbivudine (Tyzeka) Nucleoside reverse transcriptase inhibitor
2006 25% in HBeAg positive at year 2;  11% in HBeAg negative at year 2
Tenofovir (Viread) Nucleoside Reverse transcriptase inhibitor 2006
0% at year 2; adefovir resistant HBV should be treated with tenofovir and another HBV antiviral




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