My Periodic Table

Oliver Sacks, shining brilliantly as always:

“Next to the circle of lead on my table is the land of bismuth: naturally occurring bismuth from Australia; little limousine-shaped ingots of bismuth from a mine in Bolivia; bismuth slowly cooled from a melt to form beautiful iridescent crystals terraced like a Hopi village; and, in a nod to Euclid and the beauty of geometry, a cylinder and a sphere made of bismuth.”

bismuth

Of Mice and Men: The Basic Science of Tuberculosis

Today, some basic basic science. The August 1 issue of the Journal of Infectious Diseases has two interesting articles which use mice to investigate the pathogenesis of TB, hence, the John Steinbeck title. Hopefully we’ll all learn something along the way!

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The first article, written by researchers from Seattle, is entitled “Interferon γ and Tumor Necrosis Factor Are Not Essential Parameters of CD4+ T-Cell Responses for Vaccine Control of Tuberculosis.”  Mice and cytokines, yikes (eyes glazing over)– I definitely didn’t take “Mouse Immunology 101″ during medical school.

So the world desperately needs a new TB vaccine. The BCG vaccine was developed by Albert Calmette and Camille Guérin almost 100 years ago. It has some efficacy and is still used widely but clearly isn’t good enough to stop TB transmission.

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In addition, MDR-TB and XDR-TB are a growing threat, as I’ve described elsewhere on this blog. Unfortunately, a 2013 Lancet clinical trial of the MVA85A vaccine in South African infants did not show efficacy against TB. That MVA85A vaccine was a subunit boosting vaccine and was designed to enhance whatever protection is already provided by BCG, but it didn’t work. 

Here’s the “big picture” as I understand it. The adaptive immune system evolved to protect us from microbes. CD4+ T cells mostly act by producing cytokines that communicate with other cells of the innate or adaptive immune systems. CD4+ T cells can be subdivided into several subgroups based on the cytokines they produce (i.e. TH1, TH2, and TH17).

CD4+ T cells, interferon γ (IFN-γ), and tumor necrosis factor (TNF) are thought to be essential for the control of TB. Recall that activated antigen presenting cells (APCs) make cytokines that influence the type of T helper cell that is produced. For example, in the figure below, an APC gets this CD4+ T cell jazzed up to become an effector T cell (Th1 cell) and that pumps out IFN-γ. Then, the macrophage becomes activated and gets better at killing the bacteria. IFN-γ also stimulates macrophages to produce more IL-12, which in turn potentiates TH1 cell development (setting up a positive feedback loop).  But TB is one tough cookie. After infecting the macrophages, it is thought to kill them, leading to caseous necrosis (more on this later).

IFN-γ is essential to prevent progressive, fatal infection with TB. Clinically, we frequently use interferon gamma release assays (IGRAS), such as TSPOT and Quantiferon TB Gold, in the place of skin testing (PPD/TST). IGRAS measure T cell release of IFN-γ following stimulation by antigens unique to TB.

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What about TNF? It’s another cytokine that is synthesized by activated macrophages and T cells and serves to activate other cells in the immune system. But TNF blockers (like infliximab) cause can cause people with latent TB to reactivate and develop active TB. I’ve seen it happen and it isn’t pretty.

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Ok, back to the first JID article. The activity of TH1 cells that simultaneously produce IFN-γ and TNF has been proposed as a candidate mechanism of vaccine efficacy. But the failed MVA85A vaccine trial means we need to look more closely at this. So the authors used a mouse model of T-cell transfer and aerosolized TB infection to assess the contributions of TNF and IFN-γ to vaccine efficacy. To do that, they gave mice a vaccine called “ID93+GLA-SE” (who knew you could vaccinate mice for TB!) The ID93 vaccine apparently elicits a high frequency of multifunctional TH1 cells and reduces pulmonary TB by approximately 90% in vaccinated mice (i.e. it limits TB infection).  After immunization with the ID93 vaccine, the researchers isolated T cells from the donor mice and transferred them intravenously into naive, uninfected recipients. Then, mice were infected with M. tuberculosis H37Rv and assessed for bacterial burdens.

But what is H37Rv? It is a strain of tuberculosis which was originally isolated from human lungs in 1905 by Dr. Edward R. Baldwin. H37 originally gained attention for its virulence in the guinea pig model. Nowadays, you can purchase H37Rv from an organization called ATCC which is located in Manassas, Virginia. 110 years later, it is still used widely in scientific experiments. More on H37Rv later.

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Now on to results from the paper. As seen in this graph below, mice were not protected by vaccination with ID93, indicating that CD4+ T cells are necessary to transfer protection against aerosolized TB. However, neither CD4+ T cell–produced TNF nor host cell responsiveness to IFN-γ were necessary for protection. The authors’ conclusion is that induction of TH1 cells that coexpress IFN-γ and TNF is not a requirement for vaccine efficacy against TB.  However, IFN-γ and TNF are essential for control of TB in nonvaccinated animals.

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What did I take away from this first paper, besides that fact that I clearly need to take “Mouse Immunology 101″ and we aren’t getting an effective TB vaccine anytime soon?  I learned that CD4+ T cells are necessary and sufficient for vaccine efficacy. But IFN-γ and TNF are not needed for vaccine-elicited control of aerosolized TB. Therefore, induction of TH1 cells is not needed for the generation of protective immunity against TB by vaccination. More basic science studies are needed before additional TB vaccine trials are launched.

Now, on to the second JID study, by researchers in the UK, entitled “The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.” This study I found a lot easier to follow, perhaps because their article was full of color figures and photos (not just black and white bar graphs)! The British researchers are questioning a central tenet of TB pathogenesis, that caseous necrosis leads to extracellular matrix destruction (Figure A, below). When we say “caseous” necrosis, we mean cheeselike (human tissue, destroyed by TB, looks like cheese). In this set of experiments, the researchers infected mice with TB (the H37Rv strain used in the Seattle article and a more recently isolated strain of TB). They concluded that collagen / extracellular matrix destruction happens first, leading to cell death and caseous necrosis later (see Figure B).

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Surprisingly, the British researchers found that the H37Rv strain of tuberculosis did not cause caseous necrosis or formation of multinucleate giant cells in infected mice. However, these pathologies were observed after infection with a recently isolated clinical strain of M. tuberculosis. The authors write:

“This implies that the prolonged laboratory culture of H37Rv since its isolation from a patient in 1905 has resulted in loss of currently unidentified factors that cause giant cell formation and caseous necrosis despite being able to proliferate rapidly.”

I’m certainly not a basic scientist, but I would like to ask the Seattle researchers what they think of this. Given that the H37Rv strain may have changed significantly since 1905, is it appropriate to have been used in the Seattle study?

Dr. Tim Lahey, an infectious diseases doctor at Dartmouth responded on Twitter, “lab adaptation is a valid concern. Even greater is that mice don’t develop latency so model applicability to us is uncertain.”

Treating Hepatitis C in people living with HIV

It was a sunny San Diego day in 2010 at the UCSD Medical Center. I sat in a conference room on the 6th floor and listened to my attending physician Dr. David Wyles as he gave us an overview of the new pipeline of drugs for Hepatitis C. He said (paraphrasing here), “there are so many new medications. The protease inhibitors, NS5A inhibitors, NS5B inhibitors. Treatment is going to change dramatically in the next few years.” I remember feeling a bit overwhelmed as he described the ongoing clinical trials. Treatment of Hepatitis C was so straightforward back then in 2010 (for the doctors). All we really had was Pegylated Interferon and Ribavirin. But that was all about to change.

Now, five years later, Dr. Wyles has been proven right. He is first author of a new NEJM paper, Daclatasvir plus Sofosbuvir for HCV in Patients Coinfected with HIV-1. (Another paper that came out with it is Ledipasvir and Sofosbuvir for HCV in Patients Coinfected with HIV-1. My medical school mentor Dr. Pablo Tebas and ID fellowship attending physician Dr. Paul Sax are authors on that one). Both papers address how we can treat hepatitis C in people living with HIV. My questions about these drugs have less to do with their efficacy or safety (those issues will continue to be addressed as they are rolled out more widely). Rather, I am concerned by cost and human rights. There is a huge ongoing debate about costs of the drugs, who is able to access them, and who pays for them.  Many journalists have written about the cost of the drugs (including Abby Goodnough in today’s New York Times) but I don’t think the health equity aspects have received enough attention. Take some time to think about what the right to health means to you. Do you believe that everyone should receive high quality health care? What about “expendable” people like IV drug users? If your family member had HIV and Hepatitis C, what would you do to ensure that they were able to receive the best treatment?

Globally, an estimated 4 million to 5 million persons are chronically infected with both HIV and Hepatitis C and are at risk for life threatening diseases like liver cancer and cirrhosis. These new drugs are showing that it is possible to cure hepatitis C in people living with HIV. Do we have the societal will to ensure that they reach the people who need them the most?

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“STARTING” antiretroviral therapy as “TEMPRANO” (soon) as possible

I feel obliged to write something about the two slam-dunk NEJM studies, START and TEMPRANO, since it seems like everyone else is (official NEJM editorial by Salim Abdool Karim; START Journal Watch review by Raj Gandhi and TEMPRANO review by Carlos Del Rio; numerous commenters on Facebook and Twitter). Suffice it to say that momentum is growing rapidly towards immediate ART for all, irrespective of CD4 count. However, as Karim notes, there are still 36.9 million people living with HIV and “only” 15 million receiving ART (I say only in quotes because a decade ago no one would have predicted we would have 15 million people on HIV therapy in 2015).

Exciting, but I am not convinced that the money or political will is there to get all 36.9 million people on ART, properly. HIV is not a disease where you can hand someone a pill, check them off your list, and wave goodbye. Proper HIV therapy requires trained clinicians, social support, medication supply chain, laboratory (especially viral load!), and additional resources. And what about Isoniazid Preventive Therapy (IPT)? It’s one thing to run a big clinical trial in 9 care centers in the capital city of one country. It’s a very different challenge altogether to scale up IPT across a continent and around the world. Without significant new resources in the global fight against TB, there is a very real risk of people with active TB receiving IPT monotherapy and adverse events occurring. We need a ground-swelling of support from concerned individuals if everyone living with HIV is to gain access to lifesaving therapy. This is our moral obligation.

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HIV Incidence and Spatial Clustering in Mozambique

A sobering paper from Manhiça, Mozambique just came out. I have not spent significant time in Manhiça but have passed through before (often buying cashews from roadside vendors). Manhiça is a small town on a highway a couple of hours north of Maputo. It has extremely high HIV prevalence (almost 40% in adults). The Centro de Investigação em Saúde de Manhiça (CISM) is a research center associated with the Spanish government. CISM conducts a number of research studies on malaria and HIV and since 1996 has been running a demographic surveillance system. In this study, the researchers estimated overall HIV incidence to be 3.6 new infections per 100 person-years at risk. They used a Spatial Scan Statistics program to identify areas with disproportionate excess in HIV prevalence. They found a cluster of high HIV prevalence near a sugar mill in Manhiça and argue that it might be a “hot spot” related to migration. However, we do not know that HIV infections actually occurred in this geographic area.

To stop HIV transmission, we must reduce HIV incidence (i.e. new HIV infections). The most effective method of doing that is getting everyone on antiretroviral therapy with an undetectable HIV viral load.  Once the viral load is undetectable, HIV transmission virtually ceases, as we learned from HPTN-052. The question is how to target ART to people most likely to transmit the HIV virus, and make sure all people have an undetectable viral load, all in an era of dwindling financial resources. Even in the United States, this is a complex undertaking, as Jon Cohen recently described in Science.

My question for the Manhiça researchers is ART coverage. Many people have been working extremely hard in Mozambique to increase access to lifesaving ART. The authors state that participants in their study with an HIV positive result were offered medical follow up at the Manhiça outpatient clinic, which included CD4 counts, clinical management and provision of ART according to national guidelines. However, we do not know what percentage of HIV positive individuals actually got on ART and achieved an undetectable viral load. As far as I know, viral load is rarely done in Manhiça. Achieving virologic suppression across a population is an enormous task (we certainly haven’t been able to do it in the United States). Manhiça has more resources than most districts in rural Mozambique but expanding access to ART remains a major challenge. Yet this is an issue well beyond drugs and money. As we saw in West Africa with Ebola, it has to do with human resources, training, and community partnerships. It would be interesting to see a qualitative study from people in Manhiça living with HIV, health workers, and community members regarding those issues.

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Linezolid and Delamanid for XDR-TB

Drug-resistant TB is a global crisis. Treatment is prolonged and poorly tolerated because of side effects. In addition, research on new regimens has been slow. Today’s NEJM has two letters regarding treatment for extensively drug-resistant tuberculosis (XDR-TB).

First, a letter from South Korea, regarding final study outcomes of a previously published clinical trial of Linezolid. Interestingly, 27 of 38 patients (71%) with chronic XDR-TB were cured of the infection. Adverse events were frequent and many of the patients required a dose reduction from 600 to 300 mg daily but most were able to tolerate two years of treatment. Resistance was found in 11% of patients. Beyond efficacy, safety, and resistance, I wonder about the cost of linezolid. Linezolid costs $364/day in the United States. Unless Pfizer is going to provide a major discount, two years of linezolid would come to to $265,720 per patient, atop the other costs of XDR-TB treatment. That’s not affordable for any tuberculosis program I’m aware of. Finally, the authors state that newer oxazolidinones (i.e. tidezolid) might be effective in XDR-TB. Research on newer oxazolidinones for XDR-TB has not been conducted yet, but my understanding is the skin/soft tissue infection trials that got tidezolid approved only gave 6 days of drug (3 weeks outside the trials). Will 2 years of tidezolid be safe and tolerable? No one has any idea.

Second, a letter from Otsuka researchers regarding Delamanid. In “Trial 204″ of delamanid, the primary end point was 2-month sputum-culture conversion, which was defined as a negative culture for 5 consecutive weeks. I wonder about 2-month sputum-culture conversion as a surrogate end point. Will 2-month conversion really correlate with durable cure of XDR-TB? And the number of patients here is so small, it is hard to know what to make of these data.

My friend Anthony Cannella from the University of Florida replied to my post. He said that cost of drugs and resistance will remain major issues in the fight against drug-resistant TB. We really need a T-cell vaccine for TB. I couldn’t agree more.

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Recent transmission of TB in China

Drug-resistant tuberculosis (DR-TB) is a dreadful disease. It is extremely difficult to cure, often requiring 2 years of toxic medications. With >1.3 billion people in China, there are clearly many cases of DR-TB each year. Questions include exactly how many cases, where are they occurring, and what can be done to reduce DR-TB transmission? Genotyping technology helps epidemiologists understand the spread of infectious diseases in ways that were impossible a few years ago. In this blog post, I will review a recent CID research paper by Yang et al that uses VNTR genotyping to investigate transmission of TB in China.

First, a bit about tuberculosis. TB occurs along a spectrum from latent TB infection (LTBI) to symptomatic active disease. I describe LTBI to my patients as “sleeping TB.” The reason is that LTBI is taking a nap in your body but it can wake up at any time and make you very sick with active TB (coughing, weight loss, etc). That’s the reason we give months of chemoprophylaxis to LTBI patients (for example, people with a positive PPD/TST or IGRA).

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In China, there are thought to be approximately 1 million new cases of active TB each year, but the true number of cases might be much greater because of difficulties of TB surveillance in such a large country, and a reliance on the AFB smear. AFB smear has been used for 120 years and has a sensitivity much lower than TB culture. There are though to be >60,000 cases of  multi-drug resistant tuberculosis (MDR-TB) in China each year, but that number may be an underestimate because most cases of TB are not cultured and do not have drug susceptibility testing (DST) performed. Even when culture is performed in China, it seems to be done most frequently on Lowenstein-Jensen solid agar (LJ) which is not as sensitive as liquid culture.

In this CID paper, you will read that Yang and colleagues performed a population-based molecular epidemologic study in 5 study sites (in 5 provinces in China) over three years (2009-2012). The authors identified 2274 culture-positive TB patients over three years. Just hearing those numbers made me pause. China has over 1 million new cases of active TB each year.  Are these 2274 cases representative of the enormous burden of TB across this vast country? Unlikely, if you know anything about sampling frames (although the authors don’t argue that their study is nationally representative.  A nationally representative study would have been incredibly expensive).

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Now, on to the laboratory methods. Yang et al used variable number tandem repeats (VNTR) typing which was optimized for the TB genotypes found in China. VNTR typing is a “conventional genotyping method” and is thought to have better resolution than older methods like RFLP or spoligotyping. VNTR allowed Yang et al to identify the “Beijing genotype,” the most prevalent family of TB in China. However, VNTR cannot detect genetic variations to the granular level of whole-genome sequencing (WGS). When you look at the most recent papers published in molecular TB epidemiology, most of them are using WGS (for example this paper from Switzerland and this one from California). WGS allows a higher resolution and helps researchers identify TB transmission hotspots and missing links in transmission chains. In the future, we will likely be reading papers from China which use WGS, just not this study.

Of the 2274 TB cases, using VNTR, Yang found that 705 (31%) had clustered isolates, whereas 1569 had unique isolates. This led them to conclude that recent transmission contributes significantly to the TB burden, as one in three TB patients was a secondary case due to recent transmission. In an accompanying editorial, Arend and van Soolingen explore this finding, explaining that 31% may underestimate the true percentage of clustered cases. (Yang relied on passive case finding, children were excluded, the study’s time frame was only 3 years, and migration may have led to an underestimation). Arend and von Soolingen ask the rhetorical question if 31% of clustered cases might represent the tip of the iceberg regarding recent TB transmission in China. It’s impossible to say based on this study but I suspect the answer is yes.

With such a large percentage of recent transmission, the implication is that China should focus on rapid molecular diagnosis and appropriate treatment of active TB, including DR-TB (i.e., FAST). This represents a major operational challenge in such an huge country. The cost of Xpert MTB-RIF alone (or other molecular TB tests) would represent a huge financial burden, along with the cost of drugs, programmatic support, etc.

TB clinicians often think of DR-TB as having reduced transmissibility when compared to drug-sensitive TB. However, Yang found that MDR-TB bacteria seemed to be more transmissible than drug-sensitive TB. This could be related to the genomic aspects of the Beijing genotype or MDR-TB in China, but much more research is needed. More transmissible or less, DR-TB should have our attention. DR-TB is here to stay, in China and throughout the world. We cannot afford to be complacent, as the study by Yang et al makes clear.

References:

Editor’s ChoiceTransmission of Mycobacterium tuberculosis in China: A Population-Based Molecular Epidemiologic Study 

Editor’s ChoiceEditorial Commentary: Genotyping of Mycobacterium tuberculosis in China and Missing Links in the Chain of Ongoing Transmission of Tuberculosis