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Posts Tagged ‘Aspergillus fumigatus’


Allogeneic Stem Cell Transplantation

Writer and Curator: Larry H. Bernstein, MD, FCAP

This article has the following structure:

9.3.1  Cell based immunotherapy

9.3.2  Photodynamic therapy (PDT)

9.3.3  Small molecules targeted therapy drugs; Tyrosine kinase inhibitors; imatinib (Gleevec/Glivec) and gefitinib (Iressa).

9.3.4 Graft versus Host Disease

9.3.5 Aspergillus Complicating Allogeneic Transplantation

Introduction

9.3.1 Allogeneic Stem Cell Treatment

http://www.lls.org/treatment/types-of-treatment/stem-cell-transplantation/allogeneic-stem-cell-transplantation

Allogeneic stem cell transplantation involves transferring the stem cells from a healthy person (the donor) to your body after high-intensity chemotherapy or radiation.

Allogeneic stem cell transplantation is used to cure some patients who:

  • Are at high risk of relapse
  • Don’t respond fully to treatment
  • Relapse after prior successful treatment

Allogeneic stem cell transplantation can be a high-risk procedure. The high-conditioning regimens are meant to severely or completely impair your ability to make stem cells and you will likely experience side effects during the days you receive high-dose conditioning radiation or chemotherapy. The goals of high-conditioning therapy are to:

treat the remaining cancer cells intensively, thereby making a cancer recurrence less likely
inactivate the immune system to reduce the chance of stem cell graft rejection
enable donor cells to travel to the marrow (engraftment), produce blood cells and bring about graft versus tumor effect

Possible Adverse Effects

The immune system and the blood system are closely linked and can’t be separated from each other. Because of this, allogeneic transplantation means that not only the donor’s blood system but also his or her immune system is transferred. As a result, these adverse effects are possible:

  • Immune rejection of the donated stem cells by the recipient (host-versus-graft effect)
  • Immune reaction by the donor cells against the recipient’s tissues (graft-versus-host disease [GVHD])

The immune reaction, or GVHD, is treated by administering drugs to the patient after the transplant that reduce the ability of the donated immune cells to attack and injure the patient’s tissues. See Graft Versus Host Disease.

Allogeneic stem cell transplants for patients who are older or have overall poor health are relatively uncommon. This is because the pre-transplant conditioning therapy is generally not well tolerated by such patients, especially those with poorly functioning internal organs. However, reduced intensity allogeneic stem cell transplants may be an appropriate treatment for some older or sicker patients.

T-Lymphocyte Depletion

One goal of allogeneic stem cell transplant is to cause the T lymphocytes in the donor’s blood or marrow to take hold (engraft) and grow in the patient’s marrow. Sometimes the T lymphocytes attack the cancer cells. When this happens, it’s called graft versus tumor (GVT) effect (also called graft versus cancer effect). The attack makes it less likely that the disease will return. This effect is more common in myeloid leukemias than it is in other blood cancers.

Unfortunately, T lymphocytes are the same cells that cause graft versus host disease (GVHD). Because of this serious and sometimes life-threatening side effect, doctors in certain cases want to decrease the number of T lymphocytes to be infused with the stem cells. This procedure, called T-lymphocyte depletion, is currently being studied by researchers. The technique involves treating the stem cells collected for transplant with agents that reduce the number of T lymphocytes.

The aim of T-lymphocyte depletion is to lessen GVHD’s incidence and severity. However, it can also cause increased rates of graft rejection, a decreased GVT effect and a slower immune recovery. Doctors must be careful about the number of T lymphocytes removed when using this technique.

Stem Cell Selection

Stem cell selection is another technique being studied in clinical trials that can reduce the number of T lymphocytes that a patient receives. Because of specific features on the outer coat of stem cells, doctors can selectively remove stem cells from a cell mixture. This technique produces a large number of stem cells and fewer other cells, including T lymphocytes.

9.3.2 Defining Characteristics of  Stem Cells

http://stemcells.nih.gov/info/basics/pages/basics1.aspx

Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic “somatic” or “adult” stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos more than 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures.

When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be “reprogrammed” genetically to assume a stem cell-like state. This new type of stem cell is called induced pluripotent stem cells (iPSCs).

Stem cells differ from other kinds of cells in the body. All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.

Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells—which do not normally replicate themselves—stem cells may replicate many times, or proliferate. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.

Scientists are trying to understand two fundamental properties of stem cells that relate to their long-term self-renewal:

  1. Why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most adult stem cells cannot; and
  2. What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?

Discovering the answers to these questions may make it possible to understand how cell proliferation is regulated during normal embryonic development or during the abnormal cell division that leads to cancer.

Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry oxygen molecules through the bloodstream (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step. Scientists are just beginning to understand the signals inside and outside cells that trigger each step of the differentiation process. The internal signals are controlled by a cell’s genes, which are interspersed across long strands of DNA and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. The interaction of signals during differentiation causes the cell’s DNA to acquire epigenetic marks that restrict DNA expression in the cell and can be passed on through cell division.

Adult stem cells typically generate the cell types of the tissue in which they reside. For example, a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of blood cells. It is generally accepted that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—cannot give rise to the cells of a very different tissue, such as nerve cells in the brain.

Through years of experimentation, scientists have established some basic protocols or “recipes” for the directed differentiation of embryonic stem cells into some specific cell types (Figure 1). (For additional examples of directed differentiation of embryonic stem cells, refer to the NIH stem cell report available at

http://stemcells.nih.gov/info/scireport/pages/2006report.aspx.)

stem cell differentiation figure1_sm

stem cell differentiation figure1_sm

http://stemcells.nih.gov/StaticResources/images/figure1_sm.jpg

9.3.3 Types of Stem Cell Transplants for Treating Cancer

http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/bonemarrowandperipheralbloodstemcelltransplant/stem-cell-transplant-types-of-transplant

In a typical stem cell transplant for cancer very high doses of chemo are used, often along with radiation therapy, to try to destroy all the cancer cells. This treatment also kills the stem cells in the bone marrow. Soon after treatment, stem cells are given to replace those that were destroyed. These stem cells are given into a vein, much like a blood transfusion. Over time they settle in the bone marrow and begin to grow and make healthy blood cells. This process is called engraftment.

There are 3 basic types of transplants. They are named based on who gives the stem cells.

  • Autologous (aw-tahl-uh-gus)—the cells come from you
  • Allogeneic (al-o-jen-NEE-ick or al-o-jen-NAY-ick)—the cells come from a matched related or unrelated donor
  • Syngeneic (sin-jen-NEE-ick or sin-jen-NAY-ick)—the cells come from your identical twin or triplet
hematopoietic stem cell transplant

hematopoietic stem cell transplant

Autologous stem cell transplants

These stem cells come from you alone. In this type of transplant, your stem cells are taken before you get cancer treatment that destroys them. Your stem cells are removed, or harvested, from either your bone marrow or your blood and then frozen. To find out more about that process, please see the section “What’s it like to donate stem cells?” After you get high doses of chemo and/or radiation the stem cells are thawed and given back to you.

One advantage of autologous stem cell transplant is that you are getting your own cells back. When you donate your own stem cells you don’t have to worry about the graft attacking your body (graft-versus-host disease) or about getting a new infection from another person. But there can still be graft failure, and autologous transplants can’t produce the “graft-versus-cancer” effect.

This kind of transplant is mainly used to treat certain leukemias, lymphomas, and multiple myeloma. It’s sometimes used for other cancers, like testicular cancer and neuroblastoma, and certain cancers in children.

Getting rid of cancer cells in autologous transplants

A possible disadvantage of an autologous transplant is that cancer cells may be picked up along with the stem cells and then put back into your body later. Another disadvantage is that your immune system is still the same as before when your stem cells engraft. The cancer cells were able to grow despite your immune cells before, and may be able to do so again. The need to remove cancer cells from transplants or transplant patients and the best way to do it is being researched.

Doing 2 autologous transplants in a row is known as a tandem transplant or a double autologous transplant. In this type of transplant, the patient gets 2 courses of high-dose chemo, each followed by a transplant of their own stem cells. All of the stem cells needed are collected before the first high-dose chemo treatment, and half of them are used for each transplant. Most often both courses of chemo are given within 6 months, with the second one given after the patient recovers from the first one.

Allogeneic stem cell transplants

In the most common type of allogeneic transplant, the stem cells come from a donor whose tissue type closely matches the patient’s. (This is discussed later under “HLA matching” in the section called “ Donor matching for allogeneic transplant.”) The best donor is a close family member, usually a brother or sister. If you do not have a good match in your family, a donor might be found in the general public through a national registry. This is sometimes called a MUD (matched unrelated donortransplant. Transplants with a MUD are usually riskier than those with a relative who is a good match.

Blood taken from the placenta and umbilical cord of newborns is a newer source of stem cells for allogeneic transplant. Called cord blood, this small volume of blood has a high number of stem cells that tend to multiply quickly. But the number of stem cells in a unit of cord blood is often too low for large adults, so this source of stem cells is limited to small adults and children. Doctors are now looking at different ways to use cord blood for transplant in larger adults, such as using cord blood from 2 donors.

Pros of allogeneic stem cell transplant: The donor stem cells make their own immune cells, which could help destroy any cancer cells that remain after high-dose treatment. This is called the graft-versus-cancer effect. Other advantages are that the donor can often be asked to donate more stem cells or even white blood cells if needed, and stem cells from healthy donors are free of cancer cells.

Cons to allogeneic stem cell transplants: The transplant, also known as the graft, might not take — that is, the donor cells could die or be destroyed by the patient’s body before settling in the bone marrow. Another risk is that the immune cells from the donor may not just attack the cancer cells – they could attack healthy cells in the patient’s body. This is called graft-versus-host disease (described in the section called “Problems that may come up shortly after transplant”). There is also a very small risk of certain infections from the donor cells, even though donors are tested before they donate. A higher risk comes from infections you have had, and which your immune system has under control. These infections often surface after allogeneic transplant because your immune system is held in check (suppressed) by medicines called immunosuppressive drugs. These infections can cause serious problems and even death.

Allogeneic transplant is most often used to treat certain types of leukemia, lymphomas, multiple myeloma,myelodysplastic syndrome, and other bone marrow disorders such as aplastic anemia.

Mini transplants (non-myeloablative transplants)

For some people, age or certain health conditions make it more risky to wipe out all of their bone marrow before a transplant. For those people, doctors can use a type of allogeneic transplant that’s sometimes called a mini-transplant. Compared with a standard allogeneic transplant, this one uses less chemo and/or radiation to get the patient ready for the transplant. Your doctor might refer to it as a non-myeloablative transplant or mention reduced-intensity conditioning (RIC). The idea here is to kill some of the cancer cells along with some of the bone marrow, and suppress the immune system just enough to allow donor stem cells to settle in the bone marrow.

Unlike the standard allogeneic transplant, cells from both the donor and the patient exist together in the patient’s body for some time after a mini-transplant. But slowly, over the course of months, the donor cells take over the bone marrow and replace the patient’s own bone marrow cells. These new cells can then develop an immune response to the cancer and help kill off the patient’s cancer cells — the graft-versus-cancer effect.

Syngeneic stem cell transplants – for those with an identical sibling

This is a special kind of allogeneic transplant that can only be used when the recipient has an identical sibling (twin or triplet) who can donate — someone who will have the same tissue type. An advantage of syngeneic stem cell transplant is that graft-versus-host disease will not be a problem. There are no cancer cells in the transplant, either, as there would be in an autologous transplant.

A disadvantage is that because the new immune system is so much like the recipient’s immune system, there is no graft-versus-cancer effect, either. Every effort must be made to destroy all the cancer cells before the transplant is done to help keep the cancer from relapsing (coming back).

9.3.4 Graft versus Host Disease

http://bethematch.org/For-Patients-and-Families/Life-after-transplant/Graft-versus-host-disease–GVHD-/

Graft-versus-host disease(GVHD) occurs because of differences between the cells of your body and the donated cells and is a common side effect of an allogeneic bone marrow transplant.

An allogeneic transplant uses blood cells from a family member, unrelated donor or cord blood unit. GVHD can affect many different parts of the body including the skin, eyes, mouth, stomach, and intestines.

There are two types of GVHD:

  • Acute GVHD: Develops in the first 100 days or so after transplant but can occur later. This primarily affects the skin, stomach, intestines, and liver.
  • Chronic GVHD: Usually develops 3-6 months after transplant, but signs can appear earlier or later. If you have had or currently have acute GVHD, you are more likely to have chronic GVHD.

The severity of acute and chronic GVHD can range from mild to life-threatening.

Doctors often see mild GVHD as a good thing after an allogeneic transplant when the transplant was done for a blood cancer. It is a sign that the donor’s immune system is working to destroy any remaining cancer cells. Patients who experience some GVHD have a lower risk of the cancer returning after transplant than patients who do not develop GVHD. If the transplant was to treat a disease other than cancer disease, like aplastic anemia, then the doctor may want to treat even mild GVHD.

Graft-versus-Host Disease

JLM FerraraJE LevineP Reddy, and E Holler
Lancet. 2009 May 2; 373(9674): 1550–1561.
http://dx.doi.org:/10.1016/S0140-6736(09)60237-3

The number of allogeneic hematopoietic cell transplantations (HCT) continues to increase with more than 25,000 allogeneic transplantations performed annually. The graft-versus-leukemia / tumor (GVL) effect during allogeneic HCT effectively eradicates many hematological malignancies.1 The development of novel strategies that use donor leukocyte infusions, non-myeloablative conditioning and umbilical cord blood (UCB) transplantation have helped expand the indications for allogeneic HCT over the last several years, especially among older patients.2 Improvements in infectious prophylaxis, immunosuppressive medications, supportive care and DNA-based tissue typing have also contributed to improved outcomes after allogeneic HCT.1 Yet the major complication of allogeneic HCT, graft-versus-host disease (GVHD), remains lethal and limits the use of this important therapy.2 Given current trends, the number of transplants from unrelated donors is expected to double within the next five years, significantly increasing the population of patients with GVHD. In this seminar we review advances made in identifying the genetic risk factors and pathophysiology of this major HCT complication, as well as its prevention, diagnosis and treatment.

Etiology and Clinical Features

Fifty years ago Billingham formulated three requirements for the development of GVHD: the graft must contain immunologically competent cells; the recipient must express tissue antigens that are not present in the transplant donor; and the recipient must be incapable of mounting an effective response to eliminate the transplanted cells.3 We know now that the immunologically competent cells are T cells, and that GVHD can develop in various clinical settings when tissues containing T cells (blood products, bone marrow, and solid organs) are transferred from one person to another who is not able to eliminate those cells.45 Patients, whose immune systems are suppressed, and who receive white blood cells from another individual, are at particularly high risk for GVHD.

GVHD occurs when donor T cells respond to genetically defined proteins on host cells. The most important proteins are Human Leukocyte Antigens (HLA)267, which are highly polymorphic and are encoded by the major histocompatibility complex (MHC). Class I HLA (A, B, and C) proteins are expressed on almost all nucleated cells of the body at varying densities. Class II proteins (DR, DQ, and DP) are primarily expressed on hematopoietic cells (B cells, dendritic cells, monocytes), but their expression can be induced on many other cell types following inflammation or injury. High-resolution DNA typing of HLA genes with polymerase chain reaction (PCR)-based techniques have now largely replaced earlier methods. The incidence of acute GVHD is directly related to the degree of mismatch between HLA proteins89 and thus ideally, donors and recipients are matched at HLA-A, -B, -C, and -DRB1, (“8/8 matches”), but mismatches may be tolerated for UCB grafts (see below).1012

Non-HLA Genetics

Despite HLA identity between a patient and donor, approximately 40% of patients receiving HLA-identical grafts develop acute GVHD due to genetic differences that lie outside the HLA loci, or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY and HA-3, are expressed on all tissues and are targets for both GVHD and GVL.13 Other minor HAs, such as HA-1 and HA-2, are expressed most abundantly on hematopoietic cells (including leukemic cells) and may therefore induce a greater GVL effect with less GVHD.1314

Polymorphisms in both donors and recipients for cytokines that are involved in the classical `cytokine storm’ of GVHD (discussed below) have been implicated as risk factors for GVHD.15 Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10), Interferon-γ (IFNγ) variants have correlated with GVHD in some, but not all, studies.1618 Genetic polymorphisms of proteins involved in innate immunity, such as nucleotide oligomerization domain 2 and Keratin 18 receptors, have also been associated with GVHD.1922 Future strategies to identify the best possible transplant donor will probably incorporate both HLA and non-HLA genetic factors.

Clinical Features of Acute GVHD

Based on an early Seattle experience, acute GVHD was defined to occur prior to day 100, whereas chronic GVHD occurred after that time.2325 This definition is far from satisfactory, and a recent National Institutes of Health classification includes late-onset acute GVHD (after day 100) and an overlap syndrome with features of both acute and chronic GVHD.26 Late-onset acute GVHD and the overlap syndrome occur with greater frequency after reduced-intensity conditioning (RIC), an increasingly widespread technique (see below). As shown in Table 1, the clinical manifestations of acute GVHD occur in the skin, gastrointestinal tract and liver.27 In a comprehensive review, Martin et al found that at the onset of acute GVHD, 81% of patients had skin involvement, 54% had GI involvement, and 50% had liver involvement.23 Recent data suggest that lungs might also be targets of experimental GVHD.28

Acute GVHD Symptoms

Table 1

Pathophysiology of Acute GVHD

Two important principles are important to consider regarding the pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated but normal inflammatory mechanisms mediated by donor lymphocytes infused into the recipient where they function appropriately, given the foreign environment they encounter. Second, the recipient tissues that stimulate donor lymphocytes have usually been damaged by underlying disease, prior infections, and the transplant conditioning regimen.29 As a result, these tissues produce molecules (sometimes referred to as “danger” signals) that promote the activation and proliferation of donor immune cells.4245 Mouse models havebeen central to our identification and understanding of the pathophysiologic mechanisms of GVHD, and canine models have been critical to the development of clinically useful strategies for GVHD prophylaxis and treatment and to the development of donor leukocyte infusions.364647 Based largely on these experimental models, the development of acute GVHD can be conceptualized in three sequential steps or phases: (1) activation of the APCs; (2) donor T cell activation, proliferation, differentiation and migration; and (3) target tissue destruction (Figure 3).

Figure 3

GVHD Pathophysiology

In Phase I, the recipient conditioning regimen damages host tissues and causes release of inflammatory cytokines such as TNFα, IL-1 and IL-6. Increased levels of these cytokines leads to activation of host antigen presenting cells (APCs). In Phase II, host APCs activate mature donor cells. The subsequent proliferation and differentiation of these activated T cells produces additional effectors that mediate the tissue damage, including Cytotoxic T Lymphocytes, Natural Killer (NK) cells, TNFα and IL-1. Lipopolysaccharide (LPS) that has leaked through the damaged intestinal mucosa triggers additional TNFα production. TNFα can damage tissue directly by inducing necrosis and apoptosis in the skin and GI tract through either TNF receptors or the Fas pathway. TNFα plays a direct role in intestinal GVHD damage which further amplifies damage in the skin, liver and lung in a “cytokine storm.”

GVHD pathophysiology nihms-115970-f0003

GVHD pathophysiology nihms-115970-f0003

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735047/bin/nihms-115970-f0003.jpg

Phase I: Activation of Antigen Presenting Cells (APCs)

The first step involves the activation of APCs by the underlying disease and the HCT conditioning regimen. Damaged host tissues respond by producing “danger” signals, including proinflammatory cytokines (e.g., TNF-α), chemokines, and increased expression of adhesion molecules, MHC antigens and costimulatory molecules on host APCs.424850 A recent report demonstrated that at one week after HCT, increased levels of TNF-α receptor I, a surrogate marker for TNF-α, strongly correlated with the later development of GVHD.51 Damage to the GI tract from the conditioning is particularly important because it allows for systemic translocation of additional inflammatory stimuli such as microbial products including lipopolysaccaride (LPS) or other pathogen-associated molecular patterns that further enhance the activation of host APCs.49 The secondary lymphoid tissue in the GI tract is likely the initial site of interaction between activated APCs and donor T cells.52 These observations have led an important clinical strategy to reduce acute GVHD by reducing the intensity of the conditioning regimen. Experimental GVHD can also be reduced by manipulating distinct subsets of APCs.53,54 In addition, non-hematopoietic stem cells, such as mesenchymal stem cells or stromal cells, can reduce allogeneic T cell responses, although the mechanism for such inhibition remains unclear.2

The concept that enhanced activation of host APCs increases the risk for acute GVHD unifies a number of seemingly disparate clinical associations with that risk, such as advanced stages of malignancy, more intense transplant conditioning regimens and histories of viral infections. APCs detect infections by recognizing conserved molecular patterns that are unique to microbes, called pathogen-associated molecular patterns (PAMPs). Among the classes of receptors that recognize such patterns, the Toll-like receptors (TLR) are the best characterized.55 For example, TLR4 recognizes LPS55 and mice with mutant TLR4 receptors that do not respond to LPS cause less GVHD when used as donors.56 Other TLRs that recognize viral DNA or RNA also activate APCs and may enhance GVHD, providing a potential mechanistic basis for increased GVHD associated with viral infections such as cytomegalovirus (CMV).57

Phase II: Donor T Cell Activation

The core of the GVH reaction is Step 2, where donor T cells proliferate and differentiate in response to host APCs. The “danger” signals generated in Phase I augment this activation at least in part by increasing the expression of costimulatory molecules.58 Blockade of co-stimulatory pathways to prevent GVHD is successful in animal models, but this approach has not yet been tested in large clinical trials.2

In mouse models, where genetic differences between donor and recipient strains can be tightly controlled, CD4+ cells induce acute GVHD to MHC class II differences, and CD8+ cells induce acute GVHD to MHC class I differences.5961 In the majority of HLA-identical HCTs, both CD4+ and CD8+ subsets respond to minor histocompatibility antigens and can cause GVHD in HLA-identical HCT.

Regulatory T cells can suppress the proliferation of conventional T cells and prevent GVHD in animal models when added to donor grafts containing conventional T cells.62 In mice, the Foxp3 protein functions as a master switch in the development of regulatory T cells, which normally constitute 5% of the CD4+ T cell population.62 Regulatory T cells secrete anti-inflammatory cytokines IL-10 and Transforming Growth Factor(TGF)-β and can also act through contact-dependent inhibition of APCs.62 It is likely that the use of regulatory T cells in clinical acute GVHD will require improved techniques to identify and expand them.

Natural Killer T cell (NKT) 1.1+ subsets of both the host and donors that have been shown to modulate acute GVHD.63 Host NKT cells have been shown to suppress acute GVHD in an IL-4 dependent manner.64 A recent clinical trial of total lymphoid irradiation used as conditioning significantly reduced GVHD and enhanced host NKT cell function.65 By contrast, donor NKT cells can reduce GVHD and enhance perforin mediated GVL in an experimental model.66

Activation of immune cells results in rapid intracellular biochemical cascades that induce transcription of genes for many proteins including cytokines and their receptors. Th1 cytokines (IFN-γ, IL-2 and TNF-α) are produced in large amounts during acute GVHD. IL-2 production by donor T cells remains the principal target of many current clinical therapeutic and prophylactic approaches to GVHD, such as cyclosporine, tacrolimus and monoclonal antibodies (mAbs) directed against IL-2 and its receptor.9 But emerging data indicate an important role for IL-2 in the generation and maintenance of CD4+ CD25+ T regs, suggesting that prolonged interference with IL-2 may have an unintended consequence of preventing the development of long term tolerance after allogeneic HCT.67 IFN-γ has multiple functions and can either amplify or reduce GVHD.68,69 IFN-γ may amplify GVHD by increasing the expression of molecules such as chemokines receptors, MHC proteins, and adhesion molecules; it also increases the sensitivity of monocytes and macrophages to stimuli such as LPS and accelerates intracellular cascades in response to these stimuli.70Early polarization of donor T cells so that they secrete less IFN-γ and more IL-4 can also attenuate experimental acute GVHD.71 IFN-γ may amplify GVHD by directly damaging epithelium in the GI tract and skin and inducing immnosuppression through the induction of nitric oxide.72 By contrast, IFN-γ may suppress GVHD by hastening the apoptosis of activated donor T cells.6973. This complexity means the manipulation of IFN-γ may have diverse effects in vivo, making it a challenging target with respect to therapeutic intervention. IL-10 plays a key role in suppression of immune responses, and clinical data suggest it may regulate acute GVHD.17 TGF-β, another suppressive cytokine can suppress acute GVHD but exacerbate chronic GVHD.74 Thus the timing and duration of the secretion of any given cytokine may determine the specific effects of that cytokine on GVHD severity.

Phase III: Cellular and Inflammatory Effector Phase

The effector phase of this process is a complex cascade of both cellular mediators such as cytotoxic T lymphocytes(CTLs) and NK cells and soluble inflammatory mediators such as TNF-α, IFN-γ, IL-1 and nitric oxide.229 These soluble and cellular mediators synergize to amplify local tissue injury and further promote inflammation and target tissue destruction.

Cellular Effectors

The cellular effectors of acute GVHD are primarily CTLs and NK cells.49 CTLs that preferentially use the Fas/FasL pathway of target lysis and appear to predominate in GVHD liver damage (hepatocytes express large amounts of Fas) whereas GVHD CTLs that use the perforin /granzyme pathways are more important in the GI tract and skin.275 Chemokines direct the migration of donor T cells from lymphoid tissues to the target organs where they cause damage. Macrophage inflammatory protein-1alpha (MIP-1α) and other chemokines such as CCL2-5, CXCL2, CXCL9-11, CCL17 and CCL27 are over-expressed and enhance the homing of cellular effectors to target organs during experimental GVHD.76Expression of integrins, such as α4β7 and its ligand MadCAM-1, are also important for homing of donor T cells to Peyer’s patches during intestinal GVHD.527778

Prevention of GVHD

Based on the evidence from animal models regarding the central role of T cells in initiating GVHD, numerous clinical studies evaluating T cell depletion (TCD) as prophylaxis for GVHD were performed in the 1980’s and 1990’s. There were three principal TCD strategies: (1) negative selection of T cells ex vivo, (2) positive selection of CD34+ stem cells ex vivo; and (3) anti-T cell antibodies in vivo.83Most strategies showed a significant limitation in both acute and chronic GVHD.8488 Unfortunately, the lower incidence of severe GVHD was offset by high rates of graft failure, relapse of malignancy, infections, and Epstein-Barr virus-associated lymphoproliferative disorders. Negative selection purging strategies using various anti-T cell antibodies achieved similar long-term results regardless of the breadth of antibody specificity.8993 One large registry study demonstrated that purging strategies using antibodies with broad specificities produced inferior leukemia-free survival than standard immunosuppression in patients receiving unrelated donor transplants.94 Several studies have investigated partial T cell depletion, either by eliminating specific T cell subsets (e.g., CD8+) or by titrating the dose of T cells present in the inoculum.9597 None of these approaches, however, has convincingly demonstrated an optimal strategy that improves long-term survival.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein expressed on a broad spectrum of leukocytes including lymphocytes, monocytes, and dendritic cells. Its use in GVHD prophylaxis in a Phase II trial decreased the incidence of acute and chronic GVHD following reduced intensity transplant.98 In two prospective studies, patients who received alemtuzumab rather than methotrexate showed significantly lower rates of acute and chronic GVHD,99 but experienced more infectious complications and higher rates of relapse, so that there was no overall survival benefit. Alemtuzumab may also contribute to graft failure when used with minimal intensity conditioning regimens.100

An alternative strategy to TCD attempted to induce anergy in donor T cells by ex vivo antibody blockade of co-stimulatory pathways prior to transplantation. A small study using this approach in haploidentical HCT recipients was quite encouraging, but has not yet been replicated.101 Thus the focus of most prevention strategies remains pharmacological manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis has also been extensively tested. The best studied drugs are anti-thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations. These sera, which have high titers of polyclonal antibodies, are made by immunizing animals (horses or rabbits) to thymocytes or lymphocytes, respectively. A complicating factor in determining the role of these polyclonal sera in transplantation is the observation that even different brands of the same class of sera exert different biologic effects.102 However, the side effects of ATG/ALG infusions are common across different preparations and include fever, chills, headache, thrombocytopenia (from cross-reactivity to platelets), and, infrequently, anaphylaxis. In retrospective studies, rabbit ATG reduced the incidence of GVHD in related donor HSCT recipients without appearing to improve survival.103104 In recipients of unrelated donor HSCT, addition of ALG to standard GVHD prophylaxis effectively prevented severe GVHD, but did not result in improved survival because of increased infections.105 In a long term follow-up study, however, pretransplant ATG provided significant protection against extensive chronic GVHD and chronic lung dysfunction.106

The primary pharmacologic strategy to prevent GVHD is the inhibition of the cytoplasmic enzyme, calcineurin, that is critical for in the activation of T cells. The calcineurin inhibitors, cyclosporine and tacrolimus, have similar mechanisms of action, clinical effectiveness and toxicity profiles, including hypomagnesemia, hyperkalemia, hypertension, and nephrotoxicity.9107 Serious side effects include transplant-associated thrombotic microangiopathy (TAM) and neurotoxicity that can lead to premature discontinuation. Although clinically similar to thrombotic thrombocytopenic purpura, TAM does not reliably respond to therapeutic plasmapheresis, carries a high mortality rate, and removal of the offending agent does not always result in improvement.108 Posterior reversible encephalopathy syndrome includes mental status changes, seizures, neurological deficits and characteristic magnetic resonance imaging findings; this syndrome has been seen in 1-2% of HCT recipients receiving and calcineurin inhibitors.109 Side effects of these drugs decrease as the dose is tapered, usually two to four months after HCT.

Calcineurin inhibitors are often administered in combination with other immunosuppressants, such as methotrexate, which is given at low doses in the early post-transplant period.9107 The toxicities of methotrexate (neutropenia and mucositis) have led some investigators to replace it with mycophenolate mofetil (MMF). In one prospective randomized trial, patients who received MMF as part of GVHD prophylaxis experienced significantly less severe mucositis and more rapid neutrophil engraftment than those who received methotrexate.110 The incidence and severity of acute GVHD was similar between the two groups, but the study closed early due to superiority of the MMF arm with respect to reduced mucositis and the speed of hematopoietic engraftment. A desire for faster neutrophil engraftment has led to the use of MMF in UCB blood transplants where graft failure is a major concern.111 MMF is also often used after RIC regimens for similar reasons.112113

Sirolimus is an immunosuppressant that is structurally similar to tacrolimus but does not inhibit calcineurin. In a small Phase II trial, it showed excellent efficacy in combination with tacrolimus;114 the drug damages endothelial cells, however, and it may enhance TAM that is associated with calcineurin inhibitors.115 The combination of tacrolimus and sirolimus is currently being compared in a large randomized multi-center trial.

RIC regimens attempt to suppress the host immune system sufficiently so that donor T cells can engraft and then ablate the lympho-hematopoietic compartment of the recipient. The term “non-myeloablative” is therefore somewhat misleading. RIC regimens produce less tissue damage and lower levels of the inflammatory cytokines that are important in the initiation of GVHD pathophysiology; this effect may explain the reduced incidence of severe GVHD following RIC compared to the full intensity conditioning used in historical controls.98116 The onset of acute GVHD may be delayed after RIC until after day 100, however, and it may present simultaneously with elements of chronic GVHD (“overlap syndrome”).116120

Treatment of Acute GVHD

GVHD generally first develops in the second month after HCT, during continued treatment with calcineurin-based prophylaxis.23121 Steroids, with their potent antilymphocyte and anti-inflammatory activity, are the gold standard for treatment of GVHD. Many centers treat mild GVHD of the skin (Grade I) with topical steroids alone, but for more severe skin GVHD and any degree of visceral GVHD involvement, high-dose systemic steroids are usually initiated. Steroid therapy results in complete remission in less than half of the patients,122 and more severe GVHD is less likely to respond to treatment.123124 In a prospective randomized study, the addition of ATG to steroids as primary therapy did not increase the response rate.124 In a retrospective study, the use of ATG in patients who showed early signs of steroid-resistance was beneficial,122 but not all studies show such benefit and ATG is not standardly used because of increased infection risks.106125126.

An increasingly common treatment for GVHD is extracorporeal photopheresis (ECP). During ECP, the patient’s white blood cells are collected by apheresis, incubated with the DNA-intercalating agent, 8-methoxypsoralen, exposed to ultraviolet light (UVA), and returned to the patient. ECP is known to induce cellular apoptosis, which has strong anti-inflammatory effects in a number of systems, including prevention of rejection of solid organ grafts.127 Animal studies show that ECP reverses acute GVHD by increasing the number of regulatory T cells.128 A Phase II clinical study of steroid-dependent or steroid refractory GVHD showed resolution of GVHD in a large majority of patients, with 50% long-term survival in this very high risk group.129 Randomized multi-center studies of this approach are needed to determine its place in the management of acute GVHD.

Another interesting strategy to treat GVHD is the blockade of the inflammatory cytokine TNF-α. TNF-α can activate APCs, recruit effector cells and cause direct tissue damage.130 In animal models, TNF-α plays a central role in GVHD of the GI tract, which is central to the “cytokine storm” and plasma levels of TNFR I (a surrogate marker for TNF-α) rise in patients before the clinical manifestations of GVHD appear. 51 A recent Phase II trial of etanercept, a solubilized TNFR II, showed significant efficacy when added to systemic steroids as primary therapy for acute GVHD. Seventy percent of patients had complete resolution of all GVHD symptoms within one month, with 80% complete responses in the GI tract and the skin. The authors also showed that plasma levels of TNFR I were a significant biomarker for clinical GVHD.131

Treatment of Chronic GVHD

In contrast to acute GVHD, the pathophysiology of chronic GVHD remains poorly understood, and it is treated with a variety of immunosuppressive agents. The response of chronic GVHD to treatment is unpredictable, and mixed responses in different organs can occur in the same patient. Confounding variables such as infection and co-morbidities also make responses hard to measure. The use of corticosteroids (with or without a calcineurin inhibitor) is the standard of care, but a randomized trial of more than 300 patients with chronic GVHD found no difference between cyclosporine plus prednisone versus prednisone alone.132 Chronic immunosuppressants, especially those containing steroids, are highly toxic and result in infectious deaths. Many second line therapies have been studied, but none has achieved widespread acceptance. As mentioned above, ECP shows some promise, with significant response rates in high-risk patients. The best responses were observed in skin, liver, oral mucosa, eye, and lung.133 This observation is particularly relevant because lung GVHD has the potential to be a particularly devastating complication necessitating lung transplant as the only therapeutic option.134135

Essential Supportive Care in GVHD Patients

Meticulous supportive care is critical for patients with both acute and chronic GVHD because of the extended duration of immunosuppressive treatments and because the multiple medications required may have synergistic toxicities. Such care includes extensive infectious prophylaxis, early interventions in cases of suspected infections, and prophylaxis against non-infectious side effects of medications (See Table 3). These complications often require rapid responses to prevent serious or irreversible damage, and are best handled in close collaboration between the primary physician and the transplant specialist.

Table 3

Recommendations for Supportive Care

All patients should receive at least fluconazole as prophylaxis against fungal infections. Invasive molds, especially aspergillus, are common in patients with prolonged steroid use.136 Prophylaxis with voriconazole or posaconazole should be considered for these patients. Usual sites of infection are the lungs, sinuses, brain, skin,137 and serial galactomannan assays may aid in the early detection.138 Candida can cause lesions in the lung and spleen, which may need screening with ultrasonography. Pneumocystis is another opportunistic infection that should receive cotrimoxazol (bactrim) prophylaxis.139

Viral infections are frequent in these patients with GVHD. Cytomegalovirus causes interstitial pneumonia and gastritis. Patients who are at risk should have their blood monitored several times monthly. Techniques that directly detect virus should be performed, such as CMV PCR or pp65 antigen, and evidence of increased viral load should prompt preemptive treatment with ganciclovir or foscarnet prior to clinical manifestations of disease. Shingles is not uncommon and acyclovir prophylaxis may be beneficial.140 Patients and caregivers should receive vaccinations against influenza, and treatment with neuraminidase inhibitors is recommended in the event of influenza infection.141142

Patients with GVHD often have IgG2 and IgG4 subclass deficiencies despite normal lgG levels, making them susceptible to infections with encapsulated organisms. Treatment of severe hypogammaglobulinemia with intravenous immunoglobulin is standard in many centers,143 but the level that triggers replacement varies considerably among transplant specialists. There is little supporting evidence for the routine use of intravenous immunoglobulin as prophylaxis144 but patients should receive routine prophylaxis (penicillin or its equivalent) due to the increased risk of streptococcal sepsis.145 Pneumococcal conjugate and hemophilus influenza vaccine may provide additional protection and are also recommended for all patients, including those with chronic GVHD.139146147 The sites of any indwelling catheters should be assessed regularly and early treatment of a suspected infection initiated. Early signs or symptoms of septic shock such as shaking chills or low blood pressure requires prompt evaluation with chest X-ray and/or CT scan, blood culture and broad spectrum antibiotics because shock may progress rapidly in these patients.

9.3.5 Aspergillus Complicating Allogeneic Transplantation

Aspergillus infections in allogeneic stem cell transplant recipients: have we made any progress?

E Jantunen, V-J Anttila and T Ruutu
BMT 2002; 30(12):925-929
http://www.nature.com/bmt/journal/v30/n12/full/1703738a.html
http://dx.doi.org:/10.1038/sj.bmt.1703738

Invasive aspergillosis (IA) is common in allogeneic SCT recipients, with an incidence of 4-10%. The majority of these infections are diagnosed several months after SCT and they are frequently associated with GVHD. The diagnosis is difficult and often delayed. Established IA is notoriously difficult to treat with a death rate of 80-90%. This review summarises recent data on this problem to assess whether there has been any progress. Effective prophylactic measures are still lacking. Severe immunosuppression is the main obstacle to the success of therapy. Recent and ongoing developments in diagnostic measures and new antifungal agents may improve treatment results to some extent, but Aspergillus infections still remain a formidable problem in allogeneic transplantation. Further studies in this field will focus on the role of various cytokines and combinations of antifungal agents.

Summary

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New England Compounding Center (NECC): Tracking the Sources of Fungal Infections

Reporter: Alan F. Kaul, R.Ph., Pharm.D,, M.S., M.B.A, FCCP

The cause of the outbreak or fungal infections caused by contaminated steroids prepared by NECC has now been confirmed and treatment guidelines for those patients affected are in place.  Unfortunately, the toll in human lives and suffering cannot be rectified.  Clearly, compounding pharmacies are licensed by each state to produce products to meet individual patient needs. They are not legally licensed to manufacture drugs for mass distribution as is a pharmaceutical manufacturer that is licensed and inspected by the FDA.

The question of how to preclude further human disasters such as this is not yet resolved.  Painting all compounding pharmacies as unreliable as some have suggested does an enormous discredit to those pharmacists operating safe and reliable facilities where sterility testing meets or exceeds recommended standards. Political grandstanding also does a disservice towards working towards a viable answer. Should the Pharmacy Compounding Accreditation Board (PCAB), an organization that inspects and certifies that its members meet or exceed USP Chapter 797 standards be given deemed status like The Joint Commission or other similar accrediting organizations to accredit compounding pharmacies? Should state Boards Of Registrations in Pharmacy of Public Health Departments be funded for additional staff to monitor and inspect sterile compounding pharmacies? If so, will the additional expense be paid by the state, the compounding pharmacies, or the patients requiring the specially prepared drugs? Ultimately, the taxpayers will be required to pay for the requisite safeguards.  While the answer is still unresolved, careful though should be given to all possible options including a combination of them in moving forward.  The status quo is not an acceptable solution to meet the needs of providing safe and effective drugs to the public.

Investigations have now confirmed that NECC is the pharmacy linked to the deadly outbreak of fungal infections caused by Exserohilum rostratum, Aspergillus fumigatus, and Cladosporium species. An estimated 14,000 patients in 23 states received steroidal injections between May 21 to September 26, 2012 from lots of drugs prepared by NECC on May 21, June 29, and August 10, 2012. These three suspected lots of drugs prepared from steroids contained 17,676 doses were shipped to 75 locations. Three hundred forty-four infections including meningitis and those of the joints and 25 deaths have been attributed to the contaminated drugs.  As of October 22, 2012, there were 54 patients with CDC confirmed fungal meningitis. Of those, 52 were due to Exserohilum rostratum and one each due to Aspergillus fumigatus, and Cladosporium species.

Several hospitals including Saint Joseph Mercy Ann Arbor Hospital (Ypsilanti, MI), a Baltimore-area emergency room, Saint Thomas Hospital (Nashville, TN) independently noted patients presenting with symptoms including headaches, sensation to light, and neck stiffness, vertigo, double-vision, and loss of muscle co-ordination. In some patients, spinal taps were suggestive of meningitis and treatment was begun. However, infectious disease specialists were unable to identify the pathogen causing the infections. In late summer, across the United States, the same pattern appeared; patients with life-threating infections and an unknown cause. In North Carolina, a 77 year-old generally healthy female patient received the third of thee epidural injections for back pain. In September, she began experiencing terrible headaches. After multiple trips for medical care and being misdiagnosed with migraines and undergoing a brain scan, a family member insisted that she be hospitalized until they could diagnose her illness. A spinal tap was performed and spinal fluid was cultured. Meningitis of an unknown cause was diagnosed.

In Tennessee a man in his 50’s who initially responded to treatment for meningitis and went home returned to Vanderbilt University Medical when his infection reappeared. The patient presented visibly ill and had unintelligible speech. Dr. April Petit an infectious disease specialist ordered the laboratory to test for unusual microbes and also fungi.  The later generally is found in immunocompromised patients. The laboratory reported that the cerebrospinal fluid culture grew Aspergillus.  After again reviewing the patient’s medical history, Dr. Petit noted that the patient had received an epidural steroid injection at the Saint Thomas Outpatient Neurosurgery Center several weeks prior to the onset of his symptoms.  She contacted the Tennessee Department of Health on September 18.

The TN Department of Health contacted Saint Thomas infection prevention staff and learned that another patient who had received an epidural steroid injection at the same facility followed a similar clinical path. Saint Thomas closed its Outpatient Neurosurgery Department on September 20 and TN notified the CDC.  State health officials in TN conducted an inspection of the Saint Thomas Outpatient neurosurgery Department to try to determine the etiology of the infection. Some considerations included improper infection control procedures, contaminated equipment, and contaminated drug.

Within a few days, several more cases of rare fungal meningitis was identified that developed between July 30 and September 18 and the TN Department of Health notified the MA Department of Public Health. The patients shared four commonalties, one being that they ad received an injection of methylprednisolone acetate manufactured by NECC.  On September 25, MA state regulators requested NECC provide a list of all medical centers that had received shipments of the suspect steroid.  They learned that the three suspect lots of drugs totaling 17,676 doses had been shipped to 75 centers.

As the CDC conducted its investigation of sites that had received the drug, they learned that other cases outside of TN had occurred including North Carolina and Michigan.  The CDC issued a health advisory.  Because of the rarity of fungal meningitis, few researchers and clinicians were accustomed to dealing with it. CDC convened an expert advisory panel to develop recommended treatment guidelines.  In addition to the initial discovery of Aspergillus fumigatus, thesubsequent cases were discovered to be caused principally by the black mold, Exserohilum rostratum.  Experts concurred that while cases caused by the former fungus were rare, cases caused by the later were even rarer and treatment options were not well identified. Many effected patients were elderly and had other co-morbidities further complicating distinguishing symptoms and making the choice of pharmacotherapy with drugs often associated with serious side effects even more difficult.

Multidisciplinary teams quickly developed expertise at Saint Joseph Mercy Ann Arbor where 66 patients were being treated.  The team included the Chief Medical Officer, pharmacists, emergency room physicians, infectious disease specialists convened for daily discussions and updates.  Drug regimens for each patient were finely tuned and a special clinic was opened to assist patients in managing their disease.

As the saga continued, more patients in multiple states were identified and treated. Unfortunately, the epidemic had already taken its grim toll.

http://www.cdc.gov/hai/outbreaks/currentsituation/

http://www.fda.gov/Drugs/DrugSafety/FungalMeningitis/default.htm

The United States Food and Drug Administration (FDA) continues to reiterate that there should be follow-up with patients who meet the following three conditions:

  1. The medication used was an injectable product purchased from or produced by NECC, including an ophthalmic drug that is an injectable used in conjunction the eye surgery, or a cardioplegic solution,
  2. The medication was shipped by NECC on or after May 21, 2012, and
  3. The medication was administered on or after May 21, 2012.

On October 22, 2012, the FDA made available a list of customers (no product information available) of NECC from May 21, 2012 sorted by state which can be found at:

http://www.fda.gov/downloads/Drugs/DrugSafety/FungalMeningitis/UCM325467.pdf

On October 23, 2012, the Centers for Disease Control and Prevention (CDC) issued a an Official Health Advisory Issuance of Guidance on Management of Asymptomatic Patients Who Received Epidural or Paraspinal Injections with Contaminated Steroid Products. CDC continues to recommend against treating using antifungal prophylaxis for treating exposed asymptomatic patients without a diagnostic testing indication meningitis. They indicate that the greatest risk of developing an infection is within the first six weeks 942 days) after injection. As an increased benefit from prophylaxis has not been demonstrated from currently available data, additional monitoring of these patients should be considered.

http://emergency.cdc.gov/HAN/han00330.asp

http://bostonglobe.com/lifestyle/health-wellness/2012/10/27/doctors-piece-together-rare-cases-fungal-meningitis-uncover-outbreak/55SIHvy58Pf8lCB0yFvpHJ/story.html

Outbreak baffled doctors until they saw common cause

By  Carolyn Y. Johnson   |   G L O B E S T AF F        O C T O B E R  2 8 ,  2 0 1 2

JEFF KOWALSKY FOR THE BOSTON GLOBE

Rhonda Hall, who had a steroid injection, talked with Anurag Malani, infectious disease specialist at a

Michigan hospital.

It was Labor Day weekend when the first patients began to trickle into an Ypsilanti, Mich., hospital complaining of headaches, sensitivity to light, and neck stiffness. Laboratory tests of the patients’ spinal fluid strongly suggested meningitis and physicians started treatment.

But in a cluster of offices on the third floor, four of Saint Joseph Mercy Ann Arbor Hospital’s infectious disease specialists wrestled with a puzzle: Why couldn’t the laboratory identify the microbe causing the infection?

 Later that week and some 500 miles away, a 51­ year­ old woman developed a powerful headache radiating into her face and headed to a Baltimore ­area emergency room. She was discharged after a normal brain scan, but returned the next day with distressing symptoms: double vision, nausea, vertigo, and a loss of muscle coordination. As her condition worsened, a spinal tap provided no clues to the underlying cause.

And then in mid­ September, Dr. Robert Latham at Saint Thomas Hospital in Nashville, Tenn., found himself perplexed by the case of a woman who returned to the hospital after a treatment for meningitis stopped working. Lab tests showed signs of a raging infection, but similarly, he could not identify the culprit.

At hospitals scattered across the country, it was the horror story of the waning days of summer. Teams of physicians faced the same medical mystery — patients with life­ threatening infections with an unknown cause. There were subtle hints that they were dealing with a highly unusual illness, and astute clinicians and state and federal health officials worked to connect the dots. Ultimately, they would discover that these seemingly isolated cases were the leading edge of an outbreak of a fungal meningitis so rare that many doctors will never see a case in their lifetimes.

 The cases would quickly be linked to three batches of an injected steroid produced by a Framingham compounding pharmacy, but by that time 14,000 people in 23 states had received the injections for back and joint pain. More than 300 have fallen ill, and 25 have died.

Still immersed in treating the illness, most doctors have not had time to reflect on it. But Latham compared the initial confusion, frustration, and growing alarm to the early 1980s, before HIV had been identified as the cause of AIDS. The impact of a tainted drug could never be compared to that global epidemic, but at Saint Thomas, where 38 patients have now been treated, the medical team had the same feeling of being overwhelmed by an unknown that was bigger than anyone imagined.

 “When the HIV patients first started presenting, we were all scratching our heads, saying, ‘What in the devil is this?’ ” Latham said. “Those of us here at Saint Thomas are having an experience similar to San Francisco General in the early 1980s, when young men were walking in” with pneumonia and cancer.

This time, the patients walking in were mostly middle­age and elderly, with signs of meningitis.

The struggle for answers

Elwina Shaw of Denton, N.C., received the third of a set of epidural injections for back pain at the end of August. A vibrant 77­year­old, Shaw was generally healthy, said her daughter, Dawn Frank, aside from a little bit of knee pain and the back trouble. She wanted back surgery, but she had been steered instead toward the shots to see whether they would help.

Shaw was working in her garden one day in September when she got a terrible headache, Frank recalled. Shaw went to the doctor, and at first was told she was having migraines. But they didn’t go away. She went to the hospital for a brain scan, but it still wasn’t clear what was wrong. She was sent home, Frank said, and was told it might be a virus.

Finally, on September 25, Frank brought her mother back to the hospital, determined that doctors would not send her away until they could figure out what was wrong. Near midnight, she remembers, they did a lumbar puncture, drawing out a sample of spinal fluid.

Frank prayed it would not be bad. Shaw’s 80 ­year ­old husband, Rex, needed her. A talented seamstress, eloquent writer, and a woman of great faith, she filled their home and lives with grace and love. She never drew attention to herself, and had always embraced being a homemaker and mother.

 The test results were clear: meningitis of unknown cause. Unbeknownst to her physicians and her family, Elwina Shaw had joined the constellation of cases that were challenging doctors and wrenching families in other states.

In Michigan, patients who responded initially to treatment for meningitis returned to the hospital, worse. In Maryland, the 51­year­old woman’s spinal fluid was tested for bacterial infection and viruses ranging from West Nile to herpes as medical teams tried to treat her, according to a report published in the  Annals of Internal Medicine . Within a week and a half of being admitted to the hospital, she was brain dead. In Tennessee, doctors were struggling to figure out how to help the woman who had seemed to recover, then relapsed.

Dr. Varsha Moudgal, an infectious disease specialist at Saint Joseph Mercy Ann Arbor in Michigan, said physicians there had been mulling over several unusual aspects of their handful of cases. Some patients seemed almost too well, Moudgal said, explaining that meningitis patients with the kind of sky­high counts of immune cells and extremely low glucose levels doctors measured would typically have more symptoms, such as altered mental abilities.

“They came in and didn’t appear to be as ill as their cerebrospinal fluid picture suggested,” Moudgal said. “They were talking to us. They were sitting up.”

Others had severe symptoms but their lab tests suggested their infections were not that bad.

The doctors turned to specialists in microbiology and pathology, asking them to rack their brains for better diagnostic methods. Physicians scoured the medical literature to see whether past cases could teach them how to treat their growing cluster of patients. Dr. Anurag Malani said he heard rumbles of a case at another hospital that echoed theirs.

“We knew something was wrong, but it was hard to put a finger on it,” Malani said. “In hindsight, I think a lot of other places were feeling the same frustration.”

Meanwhile, in Tennessee, Dr. April Pettit, an infectious disease specialist at Vanderbilt University Medical Center, had been struggling with the same disturbing pattern: A man in his 50s with what appeared to be meningitis. He initially responded to treatment, went home, and then returned, the infection careening out of control.

 When he came back, she reported in the  New England Journal of Medicine this month, he was visibly ill and his speech unintelligible. Searching for answers, she told the laboratory to test for unusual microbes, such as fungi, even though such infections are quite rare, usually occurring in people with suppressed immune systems.

“On morning rounds, Dr. Pettit gets a call from the microbiology laboratory,” said Dr. William Schaffner, an infectious disease specialist at Vanderbilt who is familiar with the case. “She steps out to get the call, and she receives the information the cerebrospinal fluid has grown a fungus: aspergillus. She is dumbfounded.”

A common denominator

Pettit reviewed her patient’s history, to see whether there was anything unusual, anything that could explain why an otherwise healthy, middle­aged man with no immune system problems could have gotten such a rare type of meningitis. Several weeks earlier, she learned, he had received an epidural steroid injection at Saint Thomas Outpatient Neurosurgery Center. It was the only thing that stood out. She contacted the Tennessee Department of Health.

Dr. Marion Kainer of the health department immediately got in touch with the infection prevention staff at Saint Thomas. She told them of the man in his 50s, whose disease had followed much the same trajectory as their patient — and who had also received an injection. Latham knew his patient had also gotten an epidural injection at the hospital’s neurosurgery clinic, but previously he had no reason to connect it to her symptoms.

“The fact we had two people with strange presentations, related to the epidural injection, I hope would have been a bellwether for us,” Latham said. But that day, they got an even clearer message that something larger was going on: Another person had been admitted with similar symptoms. That person had also had an injection at the same place.

Saint Thomas closed its Outpatient Neurosurgery Center on Thursday, Sept. 20, and Tennessee notified the Centers for Disease Control and Prevention in Atlanta. Latham accompanied state health officials on an inspection of the facility to see whether there were any clues as to where the infection had come from: Did the clinic have the proper infection ­control policies and procedures? Was there a chance equipment had been contaminated? Could it have been a contaminated drug?

 By that Sunday, other probable cases had been identified in Tennessee, and the next day the Tennessee Department of Health contacted their counterparts in Massachusetts. Late in the evening, the Tennessee officials told the Bay State regulators of six rare fungal meningitis cases that had developed between July 30 and Sept. 18 in their state. The patients had at least four things in common: one being that they had received an injection of methylprednisolone acetate made by New England Compounding Center.

A day later, state regulators asked the owners of the Framingham compounding pharmacy to compile a list of all the medical centers that had been shipped medication from three batches of the steroid that federal officials had flagged as suspicious. The lots, prepared on May 21, June 29, and Aug. 10, the officials learned, had been shipped to 75 locations — and they contained 17,676 doses.

The next day, Sept. 26, the company voluntarily recalled the products, but there was still no firm connection between the drugs and the outbreak.

Then, physicians at the High Point Regional Health System in North Carolina, where Elwina Shaw was being treated, received a call from the CDC. The High Point Surgery Center was among the places that received doses of the drug. The agency official asked whether there were any patients with symptoms similar to the Tennessee cases, according to hospital spokeswoman Tracie Blackmon. High Point did have such a patient, the hospital confirmed.

The CDC later said in a health advisory that it was that first case outside of Tennessee that was “possibly indicating contamination of a widely distributed medication.” Frank said her family was told her mother’s case helped point the finger at the contaminated drug. “The steroid was the common denominator,” Frank said.

The doctors in Michigan began to hear news reports of what was going on in Tennessee. They began to realize the common thread was the epidural injections their patients had received at a nearby clinic.

Treating an outbreak

Pinpointing the source of the infection was only the first step. Public health officials now realized that many more people were likely to be hospitalized in the coming weeks, but they had little idea how to treat them. Fungal meningitis occurs infrequently, and the circle of researchers who study such infections is small.

 The CDC convened a panel of experts to develop advice for physicians on what symptoms to watch for, how to best treat it, and when to start antifungal medications. Complicating matters was the fact that while the initial case in Tennessee involved a fungus called Aspergillus fumigatus, the subsequent cases were mainly caused by a black mold called Exserohilum rostratum.

Cases of meningitis caused by aspergillus were rare, say specialists in fungal diseases, but cases caused by black mold were even more so, making the outbreak almost entirely untrodden medical ground. The large number of elderly victims was another challenge, because many had chronic conditions that could make it difficult to distinguish symptoms or that make them unable to tolerate the harsh drugs.

Expertise rapidly developed at the centers that were hardest hit. At Saint Joseph Mercy Ann Arbor, where 66 patients had been treated as of Friday, there was a daily 9 a.m. “huddle” of health care providers, followed by a call that drew together people from across the hospital, from the chief medical officer to pharmacists to emergency room doctors to the infectious disease specialists.

Drug regimens were fine­tuned to diminish side effects, and a special clinic was set up to help patients manage the disease.

Patients will have to take the antifungal drugs for a minimum of three months — and possibly as long as a year.

More staff were brought in to help manage the flood of people who came to be tested for meningitis. On their busiest day, 66 spinal taps were drawn; during the last month, a couple hundred have been performed, Malani said.

Three patients have died, but two fell ill before the meningitis cases were connected to a fungus.

By the time Rhonda Hall showed up at the hospital a week and a half ago, systems and procedures were in place and the pace had slowed. The 49­year­old bus driver from Brighton, Mich., was in an accident a year ago that still causes her pain. She had recently had surgery on her left ankle and got a steroid injection in her hip.

Soon after, Hall found herself clutching the side of her mattress just to get out of bed, and she realized that it wasn’t just an after­effect of the surgery. Something was wrong with her hip.

After hearing about the contaminated injections on the news, she called and learned she had gotten one of the bad shots. She was diagnosed with a bone infection.

“I was very scared in the beginning,” Hall said last week, just before going into surgery to flush out the infected joint. “Now it’s to the point . . . I want it over with so I can start healing and feeling better.”

The lessons learned by physicians came too late for Elwina Shaw. During her time in the North Carolina hospital, Shaw had two strokes, her daughter said, but she was able to write her name in cursive and walk afterward. Her family was hopeful.

But her condition worsened, and she died Friday, Oct. 19. On that day, the CDC reported that 271 people were infected, 21 deceased.

Carolyn Y. Johnson can be reached at  cjohnson@globe.com. Follow her on Twitter

@carolynyjohnson.

© 2012 THE NEW YORK TIMES COMPANY

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