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The Potential Role of Infectious Agents in Diseases of Unknown Etiology
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The Potential Role of Infectious Agents in Diseases of Unknown Etiology STEVEN M. OPAL
KEY CONCEPTS • Some infectious diseases are known to induce chronic diseases (Streptococcus pyogenes and rheumatic heart disease) and illnesses remote from the initial interaction with pathogens (e.g. hepatoma from hepatitis B or adult T cell leukemia from HTLV-1 following initial infection decades earlier). • A number of diseases of unknown cause follow epidemiologic patterns that suggest a possible infectious etiology but defy a clear link with a specific pathogen. • Chronic inflammatory states induced by micro-organisms can lead to neoplastic transformation (e.g. ‘scar’ carcinoma of the lung in areas of chronic infection such as tuberculosis or bladder carcinomas from longstanding urinary schistosomiasis) and antigenic mimicry between pathogens and host tissues can induce inflammatory lesions or vasculitis (e.g. Guillain–Barré syndrome from cross-reacting nerve sheath antiganglioside antibodies to Campylobacter jejuni outer cell membrane antigens or cross-reacting antineutrophil cytoplasmic antibodies associated with granulomatosis with polyangiitis from cell wall protein antigens expressed on some strains of Staphylococcus aureus. • Perturbations of the human microbiome are already known to be associated with clinical disease (e.g. chronic diarrhea after Clostridium difficile-associated diarrhea or changes in the microbiota in inflammatory bowel disease and irritable bowel syndrome), and it seems likely that other diseases are caused by disruption of the normal endogenous microbiota. • Increasing access to high throughput nucleic acid sequencing and metagenomic studies should be able to solve some longstanding issues concerning the role of infections and disease causation in human syndromes of unknown etiology.
Introduction Ultimately all human diseases are the result of a deleterious interaction between our genomes and the environment in which we exist. Critical components of our environment are the other living things with whom we share the biosphere. We are exposed to a multitude of microbial pathogens over a lifetime and carry with us a remarkably complex and highly variable complement of endogenous micro-organisms (the microbiota) with the potential to do us good or harm if our defenses falter. The microbiota of the average adult human is estimated to contain 1014 organisms and consists of at least 1000 different species, of which nearly half have yet to be successfully isolated and cultivated.1,2 When Pasteur, Koch and many other microbiologists of the latter half of the 19th century first confirmed the ‘germ theory’ of disease, they revolutionized medicine and the way we think about disease. The fatalistic perception of human illness as a passive, inevitable consequence of life was replaced during this age of enlightenment by the notion that at least some diseases are caused by invading microorgan-
isms and are, therefore, possibly preventable or treatable. The search for the microbial etiology of all human maladies was on, and continues to the present time.3 The most recent example is the discovery of the infectious cause of peptic ulcer disease. Generations of physicians and surgeons considered it axiomatic that ulcers were the result of excess gastric acidity (‘no acid, no ulcer’). When Marshall and Warren4 finally convinced a skeptical scientific and medical community in the 1980s that most cases of chronic gastritis and peptic ulcers were actually caused by a spiral gram-negative bacterial organism (Helicobacter pylori) inhabiting the gastric mucosa, they revolutionized the management of peptic ulcer disease.5 The genomic era in both human and microbial genetics has rekindled an interest in the discovery of microorganisms as a cause of human disease. Many chronic inflammatory, neoplastic and neurologic diseases carry tantalizing hallmarks suggestive of an underlying infectious cause.6 Are there other common, or not so common, chronic idiopathic conditions still waiting to be revealed as infectious diseases?
The Definition of Disease Causation and Etiologic Agents in the Pre-Genomic Era In 1890, Koch first formally presented his famous postulates to define the requisite evidence needed to confirm the causative role of a microorganisms in human disease (see Chapter 1).3 Yet even Koch himself recognized from the beginning that these guidelines were not relevant for all pathogens. Koch famously observed that leprosy was likely caused by the mycobacterial-like bacillus (Mycobacterium leprae) that was readily identifiable on tissue stains of involved skin, yet he (or anyone else for the next century) was never able to grow the organism on artificial media. This violated his own third postulate; nonetheless, he was comfortable assigning a pathogenic role to these readily visible leprosy bacilli. A myriad of well-known human pathogens, including many viruses, rickettsia, spirochetes and other pathogens, do not fulfill all of Koch’s postulates and yet their etiological identities are well established. The fact that many opportunistic potential pathogens colonize people for prolonged periods without causing disease (e.g. Candida albicans, Staphylococcus aureus, etc.) was not appreciated in the early days of clinical microbiology. This violates Koch’s second postulate. Moreover, many human diseases caused by well-known microbial pathogens lack comparable animal models (e.g. rheumatic fever, or progressive multifocal leukoencephalopathy) and therefore do not meet all of Koch’s postulates. The value and limitations of Koch’s postulates are described in detail in Chapter 1 in this text.
Alternative Views of Microbial Causation of Human Disease in the Genomic Era Periodontitis is among the most prevalent infectious diseases, afflicting 30–70% of adults worldwide.7 This oral infection has been extensively studied by both culture-dependent and culture-independent methods.8
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A consistent pattern of microbial pathogens has now emerged as the cause of periodontitis. The genomic evidence strongly supports the communal cause of periodontal disease. Biofilms consisting of Streptococcus mutans and other oral streptococci residing in a mono- or disaccharide-rich environment in the oral cavity express exotoxins that gradually induce dental caries over time. A synergistic combination of bacterial pathogens living within the biofilm lining the gingival crevice then gives rise to periodontal disease. A combination of Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans gradually leads to gingivitis, bone resorption and the full expression of periodontitis. Later disease progression often involves additional noncultivable bacterial strains and a methanogenic Archaea organism (the first example of a member of the Archaea kingdom associated with a human disease).1,2 Important human illnesses are the result of long-term sequelae of remote infections (the ‘hit and run’ hypothesis) that occur months or even years after the infection has cleared. Examples include Guillain– Barré syndrome following enteric infection with specific serotypes of Campylobacter spp.; Sydenham’s chorea as a late manifestation of the non-suppurative complications of S. pyogenes pharyngitis; or postinfectious reactive arthritis (formerly known as Reiter’s syndrome) following enteric infections. The pathogen is usually no longer cultivable from these patients when the disease is manifest and yet their immunologic footprint clearly identifies the etiologic organism in each case.1,6 A large number of immune-mediated, inflammatory and neoplastic diseases may have, as their proximate cause, a specific pathogen or group of micro-organisms as an inducer or essential propagator of human illness. Exposure of the susceptible host with the appropriate genotype, and the fortuitous concurrence of the necessary set of environmental and epidemiologic circumstances conspire together to initiate and maintain an aberrant host response leading to overt clinical disease. Sarcoidosis might be an example of a disease induced by environmental and generally nonpathogenic bacterial organisms in some genetically susceptible individuals.9–12 A specific genetic locus has been recently identified that appears to be linked with sarcoidosis.13 Excessive and aberrant granulomatous reactions to common microorganisms such as Propionibacterium acnes14–16 or tuberculous or environmental, non-tuberculous, mycobacterial antigens17,18 might induce sarcoidosis in these susceptible patients. Similarly, genetic susceptibility to inflammatory bowel disease is well described in association with NOD2 polymorphisms and other mutations in pattern recognition receptors (Toll-like receptor 4) resulting in defective clearance of enteric bacteria and microbial antigens and mediators along the mucosal epithelium of the small and large bowel.19–24 This genetic predisposition to inflammatory bowel disease, in concert with alterations in the normal microenvironment and enteric microbial flora, is currently one of the favored hypotheses to explain the pathogenesis of Crohn’s disease.25–27 Endemic Burkitt’s lymphoma in children in sub-Saharan Africa provides another unfortunate example of a confluence of permissive environmental conditions and collaboration between two widely disparate pathogens to cause neoplasia. Burkitt’s lymphoma is common in African children living in holo-endemic regions where mosquitoborne falciparum malaria exists. Neoplastic transformation to lymphoma is generally attributed to an aberrant host response to Epstein–Barr virus (EBV) infection. Recent evidence indicates that Plasmodium falciparum, via its major erythrocyte protein PfEMP-1, induces general immunosuppression and profound B-cell proliferation that activates productive infection with EBV. High-level replication of EBV promotes the translocation and activation of the potent oncogene c-myc resulting in malignant transformation to Burkitt’s lymphoma.28,29 The potential role of other viral, bacterial and parasitic infectious diseases in the pathogenesis of other neoplastic diseases is surveyed in Table 69-1.28–31 There are a large number of clinical settings in which a specific micro-organism or group of micro-organisms might be necessary and sufficient to cause a variety of human illnesses, yet go unnoticed when
TABLE 69-1
Microbial Disease Associations with Neoplastic Diseases Level of Evidence of Causation*
Possible Pathogen
Neoplasia
Helicobacter pylori
Gastric adenocarcinoma Gastric lymphoma
+++ ++
Salmonella typhi
Biliary carcinoma in carriers
+++
EBV
Burkitt’s lymphoma (with Plasmodium falciparum) Hodgkin’s disease Nasopharyngeal carcinoma Primary CNS lymphoma Immunoblastic lymphoma
+++
Hepatitis B
Hepatoma
+++
Hepatitis C
Hepatoma
+++
Schistosoma haematobium
Bladder carcinoma
++
Clonorchis sinensis
Cholangiocarcinoma
++
HPV-16/18
Cervical carcinoma Colorectal cancer
+++ +
Helicobacter bilis
Biliary tract cancer
+
HTLV-1
Acute T-cell leukemia/lymphoma Invasive cervical carcinoma Small cell carcinoma of the lung
HERV-K10
Testicular cancer
Kaposi’s sarcomaassociated herpesvirus (HHV-8)
Kaposi’s sarcoma Primary effusion lymphoma
++ ++ +++ ++
+++ + + + +++ ++
*Level of evidence: epidemiological link only +; some evidence of causation ++; clear evidence of causation +++. CNS, central nervous system; EBV, Epstein–Barr virus; HERV, human endogenous retrovirus; HH8-V, human herpesvirus 8; HPV, human papilloma virus; HTLV, human T-cell leukemia/lymphoma virus.
relying upon standard diagnostic techniques.1–5 A number of epidemiological features and histopathologic findings suggest a microbial cause for a variety of idiopathic diseases. Such diseases as sarcoidosis,9–11 Kawasaki disease,32 multiple sclerosis,33 type 1 diabetes mellitus,34 Kikuchi–Fujimoto disease35–37 and numerous others suggest an underlying infectious cause. Caution needs to be exercised before it can be unequivocally concluded that the implicated organism is in fact the true etiologic agent.3,6 Certain organisms could be highly associated with a specific disease entity, yet simply occupy a unique ecologic niche created by the disease itself. Streptococcus gallolyticus is highly associated with neoplastic lesions in the colonic mucosa, yet there is little evidence to suggest that this organism causes colon tumors. The organism takes the opportunity to colonize the unique microenvironment adjacent to the neoplastic tissue. A survey of potential mechanisms by which the microbial cause of disease could go unrecognized using conventional diagnostic methods is provided in Table 69-2.38–41
The Hygiene Hypothesis and the Pathogenesis of Inflammatory Diseases It should also be acknowledged that, paradoxically, heavy exposure to some elements of the microbiota, particularly at a young age, may actually protect against a number of inflammatory and allergic diseases later in life. The incidence of asthma, Crohn’s disease, diabetes and multiple sclerosis has progressively increased over the last half century in higher-income nations as vaccine-preventable, and food- and
Chapter 69 The Potential Role of Infectious Agents in Diseases of Unknown Etiology
TABLE 69-2
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Mechanisms by which Causative Microbial Pathogens Might Escape Detection
Mechanism
Examples
Comments
‘Hit and run hypothesis’
Streptococcus pyogenes – chorea minor in rheumatic fever Measles – subacute sclerosing panencephalitis
Disease manifestations induced by infection but separated from body site and in time by months, years or decades
Difficult to culture or noncultivable
Tropheryma whippleii – Whipple’s disease Bartonella henselae – cat scratch disease
Genomic studies indicate enormous numbers of microbial genomes that have yet to be successfully cultured
Commensal organisms that rarely cause disease in susceptible hosts
Sacrocystis spp. – enteritis or myositis Non-tuberculous mycobacteria – respiratory infections
Difficult or impossible to prove Koch’s postulates
Disease occurs with microbial communities
Periodontitis Irritable bowel syndrome Obesity? Bronchiectasis Bacterial vaginosis
Synergistic combinations of micro-organisms cause disease
Micro-organism induces immunologic or neoplastic alterations
KSHV (HHV-8) and Kaposi’s sarcoma Helicobacter pylori and gastric carcinoma
Microbial pathogen may serve as promoter or essential inducer
HERVs
HERV-E and systemic lupus erythematosis
Widely prevalent in human genome – possible inducer or propagator for chronic diseases
Different micro-organisms trigger illness only in genetically susceptible patients
Possibly sarcoidosis Kawasaki disease Multiple sclerosis, etc.
A variety of possible microbial pathogens may induce disease at critical time intervals in susceptible individuals
HERV, human endogenous retrovirus; HHV-8, human herpesvirus 8; KSHV, Kaposi’s sarcoma-associated herpesvirus.
TABLE 69-3
Koch’s Postulates Revisited: Microbial Causation in the Genomic Era
Modified Hill and Evan’s Criteria
Genomic Criteria
Prevalence of disease higher in patients exposed to suspect pathogen(s) than those not exposed
Nucleic acid sequence of suspect pathogen should be present in diseased tissues
Incidence of disease should be higher in patients exposed to suspect pathogen(s) than controls in prospective studies
No or few copies of nucleic acid sequences of suspect pathogen should be found in the absence of disease and in normal tissue
The disease should be temporally linked to exposure to suspect pathogen(s)
If suspect pathogen sequences predate onset of disease, the copy number of sequences should increase with onset of clinical illness (temporal relationship)
A spectrum of disease should be found after exposure from mild to severe disease (dose–response)
Copy number of suspect pathogen sequences should correlate to disease severity (gene copy – host–response relationship)
A measurable host response should occur following exposure to suspect pathogen(s)
The suspect pathogen sequences should localize by in situ hybridization to affected tissues
Experimental reproduction of disease should occur in models or human volunteers
Suspect pathogen sequences should be reproducibly found in patients with similar illnesses and in animal models (if available)
Prevention of transmission of suspect pathogen should decrease incidence of disease and eradication of pathogen should decrease the disease
Resolution of clinical illness should be accompanied by reduction or elimination of suspect pathogen sequences
The whole process should make biologic sense and fit the epidemiology of disease
The suspect pathogen determined by nucleic acid homology searches should fit the biology and pathology of genetically related micro-organisms
water-borne enteric infections have dramatically decreased.42 These inflammatory diseases are much less common in low- and middleincome countries where the incidence of childhood infection remains high. The ‘hygiene hypothesis’ suggests that improved hygiene fails to prime innate immune responses to common microbial antigens, particularly chronic geo-helminth infections, in early life. This impairs mucosal immune maturation, promotes a TH2-type cytokine response and facilitates inappropriate cellular tolerance to many common microbial antigens. The end-result is excessive and disordered inflammation to many of these environmental antigens later in childhood and adulthood with resultant increased risk of inflammatory diseases. The hygiene hypothesis seems plausible yet still lacks extensive, experimental evidence in support of this appealing notion.42-45
Culture-Independent Techniques to Detect Novel Pathogens The evolving definition of what constitutes a human pathogen has moved from a formal requirement to fulfill all of Koch’s postulates to a genomic expression of Descartes’ assertion, ‘I clone, therefore I am’ (Table 69-3). This is best evidenced by the remarkable discovery of hepatitis C as the most common cause of post-transfusion hepatitis by Choo et al. in 1989.46 After decades of concerted, yet futile, efforts to isolate the implicated virus using standard virologic techniques, they attempted to clone the genome of the unknown agent directly from the serum of a chimpanzee experimentally infected and known to contain high titers of infectious agent.
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Since it was not known if it was an RNA or DNA virus, they treated the serum with reverse transcriptase and generated a large number of sequences of amplified nucleic acids to create a comprehensive phagebased cDNA library. These clones were tested against the serum of a patient with chronic, post-transfusion hepatitis in hopes the serum would contain antibodies that recognize at least some of the expressed viral peptides. After screening nearly one million clones, the reactive peptide sequence was identified. They then showed that this was an RNAse- but not DNAse-sensitive genome and, by overlapping hybridization experiments, they confirmed that this was a positive-stranded RNA flavivirus of approximately 10 000 base pairs. The mystery of post-transfusion hepatitis was finally solved by cloning, unencumbered by the need to fulfill Koch’s postulates.3,46-48 A similar cloning strategy has now been adapted from work done with genome searches for comparative microbial ecology, environmental biology and molecular phylogeny studies.2,3 Microbial DNA sequences are isolated and amplified by polymerase chain reaction (PCR) using broad range bacterial primers for essential target sequences, such as ribosomal RNA genes. These amplified, novel bacterial sequences are then cloned in a DNA library where comparative sequence analysis is performed with highly conserved sequences from known classes of micro-organisms. The genes for 16S RNA, other essential ribosomal RNA sequences, or highly conserved enzymes are scanned for sequence homologies throughout the Bacteria and Archaea kingdoms. Sequence homologies and genomic groupings can now be ex ploited to identify shared genetic space and evolutionary distance between essentially any unknown pathogen and all identifiable, yet noncultivable, genome-characterized micro-organisms that might cause human disease. Such unbiased genome searches have been instrumental to the work of Relman, Fredricks and others3 in discovering the etiology of longstanding diseases of known microbial cause, yet not cultivable organisms such as the Whipple’s disease bacillus (Tropheryma whippleii).49,50 A genome search of the organism revealed mutations in metabolic genes and the nutritional requirements needed to successfully grow the organism on artificial media for the first time.51 Newly recognized, emerging pathogens such as the causative agents of bacillary angiomatosis (Bartonella henselae) and human monocytic ehrlichiosis (Ehrlichia chaffeensis) were first identified using a similar genomic search strategy3 (Figure 69-1). Current sequencing machinery now permits the simultaneous sequencing of short unique nucleotides from small samples of DNA without the need for preparatory cloning3 with rapid genome screens with known sequences of viruses, rickettsia and numerous other difficult-to-culture pathogens. DNA subtraction is another technique that can be used to detect and define novel microbial pathogens. This methodology was successfully employed to link a previously unknown herpes-like virus (HHV8), now known as Kaposi’s sarcoma-associated herpesvirus (KSHV), as the cause of HIV-related Kaposi’s sarcoma.2,3 The DNA sequences derived from diseased tissues and a similar set of DNA sequences from normal tissues are subjected to subtractive hybridization and PCR amplification to enrich the DNA complement of DNA sequences found in the diseased sample only. Unique sequences are then analyzed to determine if any sequence homologies exist with known pathogens. As the known genomic universe of micro-organisms continues to expand, it is likely that this and related culture-independent methods will reveal new pathogens associated with human illness.52
THE ROLE OF HUMAN ENDOGENOUS RETROVIRUSES (HERVS) IN HEALTH AND DISEASE The human genome is replete with endogenous retroviruses (HERVs, also known as retrotransposons) that have entered the human germline at various times in the evolutionary past and now occupy 8.3% of the genome.53 They maintain the basic structure of retroviruses with long terminal repeats (LTR) flanking the open reading frames for polymerase (pol), including reverse transcriptase, envelope (env) and
A genomic strategy to search for novel microbial pathogens as a cause of human disease
Isolate DNA and RNA sequences from clinical material from diseased tissues Suspect pathogen
RNA
DNA
+RT cDNA
PCR
DNA
Shotgun clone DNA amplicons into plasmid vector DNA to generate a DNA library
Sequence novel DNA segments
Screen clones with specific anti-sera if available (e.g. HCV)
Search for alignment for homology with known RNA and DNA genomes or highly conserved sequences (e.g. 16S rRNA)
Confirm
Develop oligonucleotide probes or primers and search involved tissues for in situ hybridization or in situ PCR
Attempt to culture suspect pathogen using techniques for related micro-organisms
Figure 69-1 A genomic strategy to search for novel microbial pathogens as a cause of human disease. RT, reverse transcriptase; cDNA, complementary DNA; PCR, polymerase chain reaction; HCV, hepatitis C virus.
core matrix (gag) genes along with various regulatory genes. They have accumulated loss of function point mutations in structural and regulatory genes rendering them incapable of exogenous viral production. It is possible to ‘resurrect’ some recently inherited HERVs from the human genome but this is unlikely to occur spontaneously.54 However, these endogenous retroviruses are not genetically dormant. Some HERVs have physiologic roles, including syncytiotrophoblast formation in placental development and intrinsic resistance to exogenous retrovirus infection.55 HERVs also have the potential to contribute to pathologic reactions in their hosts as well as by a number of mechanisms.53 The LTR regions are transcriptionally active and serum antibodies are detectable to env and gag proteins from a number of HERVs, indicating that these structural genes are transcribed and expressed. Transposition of HERVs can inactivate cellular genes at other loci in the human genome. Moreover, the LTRs of HERVs can have polar effects in cis and promote transcriptional activation of adjacent cellular genes. Lastly, transcriptional activators are found within HERVs that might have the capacity to activate cellular genes in trans. HERVs have been implicated in induction of malignant transformation by activating oncogenes or inactivating apoptosis-inducing genes. HERVs might contribute to immune-mediated diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE) by the generation of HERV antigens that crossreact with endogenous antigens and break tolerance to normal
Chapter 69 The Potential Role of Infectious Agents in Diseases of Unknown Etiology
autoantigens. The gag p30 of class I HERVs bear striking homology with the human 70K ribonucleoprotein U1 snRNP, a frequently recognized autoantigen in SLE.56 SLE might be induced in some patients by molecular mimicry when expressed HERV gag proteins induce antibodies that cross-react with epitopes found on the 70K RNP. HERVfacilitated epitope spreading and loss of Fas-mediated apoptosis of self reacting T-cells might further contribute to autoimmune disease progression. HERVs may collaborate with other viruses, such as EBV, to induce the synthesis of endogenous superantigen motifs encoded by HERVs.57 This process is hypothesized to contribute to the immune-mediated CNS pathology in MS. HERV-K18 has superantigenic activity in its envelope peptide; expression of this Env protein is transactivated by B cell clonal expansion by co-infection with EBV. This type of immune dysregulation with the CNS is implicated in the pathogenesis of white TABLE 69-4
629
matter lesions in MS.53,57,58 Finally, recent evidence suggests that the Env protein of HERV-W can act as a ligand for the pattern recognition receptors CD14 and TLR4.59 Activation of effector cells of the innate immune system via TLR4 with cytokine and chemokine generation might further contribute to injurious CNS inflammation in MS and other demyelinating disorders. A brief review of infectious diseases that have been associated with or cause inflammatory and vasculitic diseases is presented in Tables 69-4 and 69-5.60–67
Conclusions The number of potential pathogens that might cause disease in some susceptible human populations has expanded by an order of magnitude with the recognition of the array of noncultivable micro- organisms in our environment and even among our endogenous
Microbial Disease Associations with Inflammatory Diseases
Possible Pathogen
Disease
Level of Evidence of Causation*
Propionibacterium acnes
Sarcoidosis
+ (environmental mycobacteria also implicated)
HHV-6
Multiple sclerosis (relapsing-remitting type)
+/++ (HERV-W also implicated in association with EBV or HHV-6)
Campylobacter jejuni
Cross reacting anti-ganglioside antibodies and Guillain-Barré syndrome
+++
Mycobacterium paratuberculosis
Inflammatory bowel disease
+ (high concentrations of enteric bacteria also implicated)
HERV-W
Psoriasis
+
HERV-K18
Type I diabetes
+ (enteroviruses also implicated)
Bacterial superantigens
Kawasaki disease
+
Yersinia enterocolitica
Kikuchi–Fujimoto disease
++ (KSHV also implicated)
KSHV
Multicentric Castleman’s disease
++
Mixed enteric aerobes and anaerobes
Necrotizing enterocolitis63
+
Enteric viruses
Onset of type 1 diabetes mellitus
+
Chlamydia spp. and enteric bacteria
Ankylosing spondylitis
+
*Levels of evidence: epidemiological link only +; some evidence of causation ++; clear evidence of causation +++. EBV, Epstein–Barr virus; HERV, human endogenous retrovirus; HHV, human herpesvirus; KSHV, Kaposi’s sarcoma-associated herpesvirus.
TABLE 69-5
Microbial Disease Associations – Vasculitis
Microbial Agent
Disease
Level of Evidence of Causation*
Hepatitis B
Polyarteritis nodosa, membrano-proliferative glomerulonephritis
++/+++
Hepatitis C
Mixed cryoglobulinemia
++/+++
Staphylococcus aureus
Granulomatosis with polyangiitis (formally Wegener’s granulomatosis) Churg–Strauss eosinophilic vasculitis
++ +
EBV and/or CMV
Rheumatoid arthritis
++ (HERVs also implicated)
CMV
Atherosclerosis
+ (Chlamydia pneumoniae also implicated)
Human endogenous retrovirus-E
Systemic lupus erythematosis
+ (EBV also implicated)
Parainfluenza virus
Giant cell arteritis
+ (parvovirus B19 also implicated)
Bacterial lipopolysaccharide
Antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis
+
Helicobacter pylori
Sjögren’s syndrome, atherosclerosis
+
Propionibacterium acnes
SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis)
++
*Levels of evidence: epidemiological link only +; some evidence of causation ++; clear evidence of causation +++ CMV, cytomegalovirus; EBV, Epstein–Barr virus; HERV, human endogenous retrovirus.
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microbiota. It is possible that at least some human diseases of unknown cause will eventually be attributable to noncultured organisms. Synergistic combinations of viral, bacterial and even parasitic infections are now known to cause specific clinical disease entities, and it is likely that other disease entities are caused by communal groups of microorganisms. Aberrant host responses to common micro-organisms
might explain many inflammatory and perhaps even some neoplastic diseases. The role of human endogenous retroviruses in human disease, alone or in combination with other potential pathogens, remains a fertile area of research in the coming years. References available online at expertconsult.com.
KEY REFERENCES Fredricks D.N., Relman D.A.: Sequence-based identifica tion of microbial pathogens: a reconsideration of Koch’s postulates. Clin Microbiol Rev 1996; 9: 18-33. O’Connor S.M., Taylor C.E., Hughes J.M.: Emerging infectious determinants of chronic diseases. Emerg Infect Dis 2006; 12(7):1051-1057.
Pendergraft W.F., Preston G.A., Shah R.R., et al.: Autoimmunity is triggered by cPR-3 (105-201), a protein complementary to human autoantigen proteinase-3. Nat Med 2004; 10(1):72-79. Ridaura V.K., Faith J.J., Rey F.E., et al.: Gut Microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013; 314:1079.
Weng L., Rubin E.W., Bristow J.: Application of sequencebased methods in human microbial ecology. Genome Res 2006; 16:316-322.
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REFERENCES 1. Fredricks D.N., Relman D.A.: Infectious agents and the etiology of chronic idiopathic diseases. Curr Clin Top Infect Dis 1998; 18:180-200. 2. Weng L., Rubin E.W., Bristow J.: Application of sequence-based methods in human microbial ecology. Genome Res 2006; 16:316-322. 3. Fredricks D.N., Relman D.A.: Sequence-based identification of microbial pathogens: a reconsideration of Koch’s postulates. Clin Microbiol Rev 1996; 9:18-33. 4. Marshall B.J., Warren J.R.: Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984; 1:1311-1315. 5. Blaser M.J.: Bacteria and diseases of unknown cause. Ann Intern Med 1994; 121:144-145. 6. O’Connor S.M., Taylor C.E., Hughes J.M.: Emerging infectious determinants of chronic diseases. Emerg Infect Dis 2006; 12(7):1051-1057. 7. Tonetti M.S., D’Aiuto F., Nibali L., et al.: Treatment of periodontitis and endothelial function. N Engl J Med 2007; 356:911-920. 8. Kroes I., Lepp P.W., Relman D.A.: Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci USA 1999; 96:14547-14552. 9. Müller-Quernheim J.: Sarcoidosis: immunopathogenetic concepts and their clinical application. Eur Respir J 1998; 12:716-738. 10. Miyara M., Amoura Z., Parizot C., et al.: The immune paradox of sarcoidosis and regulatory T cells. J Exp Med 2006; 203:359-370. 11. Kelly D.M., Greene C.M., Meachery G., et al.: Endotoxin upregulates interleukin-18: potential role for gram-negative colonization in sarcoidosis. Am J Respir Crit Care Med 2005; 172:1299-1307. 12. Ezzie M.E., Crouser E.D.: Considering an infectious etiology of sarcoidosis. Clin Dermatol 2007; 25(3):259266. 13. Valentonyte R., Hampe J., Huse K., et al.: Sarcoidosis is associated with a truncating splice site mutation in BTNL2. Nat Genet 2005; 37:357-364. 14. Abe C., Iwai K., Mikami R., et al.: Frequent isolation of Propionibacterium acnes from sarcoidosis lymph nodes. Zentralbl Bakteriol Mikrobiol Hyg [A] 1984; 256:541547. 15. Hiramatsu J., Kataoka M., Nakata Y., et al.: Propionbacterium acnes DNA detected in bronchoalveolar lavage cells from patients with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 2003; 20:197-203. 16. Nishiwaki T., Yoneyama H., Eishi Y., et al.: Indigenous pulmonary Propionibacterium acnes primes the host in the development of sarcoid-like pulmonary granulomatosis in mice. Am J Pathol 2004; 165:631-639. 17. Eishi Y., Suga M., Ishige I., et al.: Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis. J Clin Microbiol 2002; 40:198-204. 18. Drake W.P., Dhason M.S., Nadaf M., et al.: Cellular recognition of Mycobacterium tuberculosis ESAT-6 and KatG peptides in systemic sarcoidosis. Infect Immun 2007; 75(1):527-530. 19. Thompson N.P., Driscoll R., Pounder R.E., et al.: Genetics versus environment in inflammatory bowel disease: results of a British twin study. BMJ 1996; 312:95-96. 20. Orholm M., Binder V., Sorensen T.I., et al.: Concordance of inflammatory bowel disease among Danish twins: results of a nationwide study. Scand J Gastroenterol 2000; 35:1075-1081. 21. Williams C.N., Kocher K., Lander E.S., et al.: Using a genome-wide scan and meta-analysis to identify a novel IBD locus and confirm previously identified IBD loci. Inflamm Bowel Dis 2002; 8:375-381. 22. Lowe A.M., Yansouni C.P., Behr M.A.: Causality and gastrointestinal infections: Koch, Hill, and Crohn’s. Lancet Infect Dis 2008; 8(11):720-726. 23. Franchimont D., Vermeire S., El Housni H., et al.: Deficient host-bacteria interactions in inflammatory bowel
disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis. Gut 2004; 53(7):987-992. 24. De Hertogh G., Aerssens J., Geboes K.P., et al.: Evidence for the involvement of infectious agents in the pathogenesis of Crohn’s disease. World J Gastroenterol 2008; 14(6):845-852. 25. Barmada M.M., Brant S.R., Nicolae D.L., et al.: A genome scan in 260 inflammatory bowel diseaseaffected relative pairs. Inflamm Bowel Dis 2004; 10:1522. 26. Swidsinski A., Ladhoff A., Pernthaler A., et al.: Mucosal flora in inflammatory bowel disease. Gastroenterol 2002; 122:44-54. 27. Sartor R.B.: Does Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease? Gut 2005; 54:896-898. 28. Chene A., Donati D., Guerreiro-Cacais A., et al.: A molecular link between malaria and Epstein–Barr virus reactivtion. PLoS Pathog 2007; 3(6):826-834. 29. Thorley-Lawson D.A., Duca K.A.: Pathogens cooperate in lymphomagenesis. Nature Med 2007; 13:906-907. 30. Janulaityte-Gunther D., Kupcinskas L., Pavilonis A., et al.: Combined serum IgG to Helicobacter pylori VacA and cagA predicts gastric cancer. FEMS Immunol Med Microbiol 2007; 50:220-225. 31. Samaras V., Rafailidis P.I., Mourtzoukou E.G., et al.: Chronic bacterial and parasitic infections and cancer: a review. J Infect Dev Ctries 2010; 4(5):267-281. 32. Burns J.C.: The riddle of Kawasaki Disease. N Engl J Med 2007; 356:659-661. 33. Cooke A., Ferraccioli G.F., Herrmann M., et al.: Induction and protection of autoimmune rheumatic diseases. The role of infections.. Clin Exp Rheumatol 2008; 26(Suppl. 48):S1-S7. 34. Cooke A.: Infection and autoimmunity. Blood Cells Mol Dis 2009; 42(2):105-107. 35. Bosch X., Guilabert A., Miquel R., et al.: Enigmatic Kikuchi–Fujimoto disease: a comprehensive review. Am J Pathol 2004; 122:141-152. 36. Cho M.S., Choi H.J., Park H.K., et al.: Questionable role of human herpesviruses in the pathogenesis of Kikuchi disease. Arch Pathol Lab Med 2007; 131(4):604-609. 37. Stéphan J.L., Jeannoël P., Chanoz J., et al.: Epstein–Barr virus-associated Kikuchi disease in two children. J Pediatr Hematol Oncol 2001; 23(4):240-243. 38. Quigley E.M.: Bacteria: a new player in gastrointestinal motility disorders – infections, bacterial overgrowth, and probiotics. Gastroenterol Clin North Am 2007; 36(3):735-748. 39. Pasarica M., Dhurandhar N.V.: Infectobesity: obesity of infectious origin. Adv Food Nutr Res 2007; 52:61-102. 40. Ridaura V.K., Faith J.J., Rey F.E., et al.: Gut Microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013; 314:1079. 41. Boyton R.J., Reynolds C.J., Quigley K.J., et al.: Immune mechanisms and the impact of the disrupted lung microbiome in chronic bacterial lung infection and bronchiectasis. Clin Exp Immunol 2013; 171(2):117123. 42. Bach J.-F.: The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 2000; 347:911-920. 43. Baumgart D.C., Sandborn W.J.: Inflammatory bowel disease: cause and immunobiology. Lancet 2007; 369:1627-1640. 44. Elson C., Cong Y., Qi F., et al.: Molecular approaches to the role of the microbiota in inflammatory bowel disease. Ann NY Acad Sci 2006; 1072:39-51. 45. Suau A., Bonnet R., Sutren M., et al.: Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 1999; 65:4799-4807.
46. Choo Q.L., Kuo G., Weiner A.L., et al.: Isolation of cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244:359-362. 47. Hill A.B.: The environment and disease: association or causation? Proc R Soc Med 1965; 58:295-300. 48. Evans A.S.: Causation and disease: the Henle–Koch postulates revisited. Yale J Biol Med 1976; 49:175-195. 49. Relman D.A., Schmidt T.M., MacDermott R.P., et al.: Identification of the uncultured bacillus of Whipple’s disease. N Engl J Med 1992; 327:293-301. 50. Fredricks D.N., Relman D.A.: Localization of Tropheryma whippelii rRNA in tissues from patients with Whipple’s disease. J Infect Dis 2001; 183:1229-1237. 51. Raoult D., Birg M.L., LaScola B., et al.: Cultivation of the bacillus of Whipple’s disease. N Engl J Med 2000; 342:620-625. 52. Dong J., Olano J.P., McBride J.W., et al.: Emerging pathogens: challenges and successes of molecular diagnostics. J Mol Diagn 2008; 10(3):185-197. 53. Colmegna I., Garry R.F.: Role of endogenous retroviruses in autoimmune diseases. Infect Dis Clin North Am 2006; 20:913-929. 54. Lee Y.N., Bieniasz P.D.: Reconstitution of an infectious human endogenous virus. PLoS Pathog 2007; 3(1):119130. 55. Mi S., Lee X., Li X., et al.: Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 2000; 403:785-789. 56. Adelman M.K., Marchalonis J.J.: Endogenous retroviruses in systemic lupus erythematosis: candidate lupus viruses. Clin Immunol 2002; 102:107-116. 57. Perron H., Garson J.A., Bedin F., et al.: Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. Proc Natl Acad Sci USA 1997; 94:7583-7588. 58. Sutkowski N., Conrad B., Thorley-Lawson D.A., et al.: Epstein–Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen. Immunity 2001; 15:579-589. 59. Rolland A., Jouvin-Marche E., Saresella M., et al.: Correlation between disease activity and in vitro cytokine production mediated by MSRV (multiple sclerosis associated retroviral) element envelope protein in patients with multiple sclerosis. J Neuroimmunol 2005; 160:195203. 60. Pendergraft W.F., Preston G.A., Shah R.R., et al.: Autoimmunity is triggered by cPR-3 (105-201), a protein complementary to human autoantigen proteinase-3. Nat Med 2004; 10(1):72-79. 61. Willcocks L.C., Lyons P.A., Rees A.J., et al.: The contribution of genetic variation and infection to the pathogenesis of ANCA-associated systemic vasculitis. Arthritis Res Ther 2010; 12(1):202. 62. Baio P., Brucato A., Buskila D., et al.: Autoimmune diseases and infections: controversial issues. Clin Exp Rheumatol 2008; 26(2 Suppl. 48):S74-S80. 63. Lin P.W., Stoll B.J.: Necrotising enterocolitis. Lancet 2006; 368:1271-1283. 64. Boman J., Hammerschlag M.R.: Chlamydia pneumoniae and artherosclerosis: critical assessment of diagnostic methods and relevance to treatment studies. Clin Microbiol Rev 2002; 15:1-20. 65. Apfalter P., Blasi F., Boman J., et al.: Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens. J Clin Microbiol 2001; 39:519524. 66. Duhaut P., Bosshard S., Ducroix J.P.: Is giant cell arteritis an infectious disease? Biological and epidemiological evidence. Presse Med 2004; 33(19 Pt 2):1403-1408. 67. Huugen D., Xiao H., van Esch A., et al.: Aggravation of anti-myeloperoxidase antibody-induced glomerulonephritis by bacterial lipopolysaccharide: role of tumor necrosis factor-alpha. Am J Pathol 2005; 167(1):47-58.
SECTION 3 Special Problems in Infectious Disease Practice: Fever
The Potential Role of Infectious Agents in Diseases of Unknown Etiology STEVEN M. OPAL
KEY CONCEPTS • Some infectious diseases are known to induce chronic diseases (Streptococcus pyogenes and rheumatic heart disease) and illnesses remote from the initial interaction with pathogens (e.g. hepatoma from hepatitis B or adult T cell leukemia from HTLV-1 following initial infection decades earlier). • A number of diseases of unknown cause follow epidemiologic patterns that suggest a possible infectious etiology but defy a clear link with a specific pathogen. • Chronic inflammatory states induced by micro-organisms can lead to neoplastic transformation (e.g. ‘scar’ carcinoma of the lung in areas of chronic infection such as tuberculosis or bladder carcinomas from longstanding urinary schistosomiasis) and antigenic mimicry between pathogens and host tissues can induce inflammatory lesions or vasculitis (e.g. Guillain–Barré syndrome from cross-reacting nerve sheath antiganglioside antibodies to Campylobacter jejuni outer cell membrane antigens or cross-reacting antineutrophil cytoplasmic antibodies associated with granulomatosis with polyangiitis from cell wall protein antigens expressed on some strains of Staphylococcus aureus. • Perturbations of the human microbiome are already known to be associated with clinical disease (e.g. chronic diarrhea after Clostridium difficile-associated diarrhea or changes in the microbiota in inflammatory bowel disease and irritable bowel syndrome), and it seems likely that other diseases are caused by disruption of the normal endogenous microbiota. • Increasing access to high throughput nucleic acid sequencing and metagenomic studies should be able to solve some longstanding issues concerning the role of infections and disease causation in human syndromes of unknown etiology.
Introduction Ultimately all human diseases are the result of a deleterious interaction between our genomes and the environment in which we exist. Critical components of our environment are the other living things with whom we share the biosphere. We are exposed to a multitude of microbial pathogens over a lifetime and carry with us a remarkably complex and highly variable complement of endogenous micro-organisms (the microbiota) with the potential to do us good or harm if our defenses falter. The microbiota of the average adult human is estimated to contain 1014 organisms and consists of at least 1000 different species, of which nearly half have yet to be successfully isolated and cultivated.1,2 When Pasteur, Koch and many other microbiologists of the latter half of the 19th century first confirmed the ‘germ theory’ of disease, they revolutionized medicine and the way we think about disease. The fatalistic perception of human illness as a passive, inevitable consequence of life was replaced during this age of enlightenment by the notion that at least some diseases are caused by invading microorgan-
isms and are, therefore, possibly preventable or treatable. The search for the microbial etiology of all human maladies was on, and continues to the present time.3 The most recent example is the discovery of the infectious cause of peptic ulcer disease. Generations of physicians and surgeons considered it axiomatic that ulcers were the result of excess gastric acidity (‘no acid, no ulcer’). When Marshall and Warren4 finally convinced a skeptical scientific and medical community in the 1980s that most cases of chronic gastritis and peptic ulcers were actually caused by a spiral gram-negative bacterial organism (Helicobacter pylori) inhabiting the gastric mucosa, they revolutionized the management of peptic ulcer disease.5 The genomic era in both human and microbial genetics has rekindled an interest in the discovery of microorganisms as a cause of human disease. Many chronic inflammatory, neoplastic and neurologic diseases carry tantalizing hallmarks suggestive of an underlying infectious cause.6 Are there other common, or not so common, chronic idiopathic conditions still waiting to be revealed as infectious diseases?
The Definition of Disease Causation and Etiologic Agents in the Pre-Genomic Era In 1890, Koch first formally presented his famous postulates to define the requisite evidence needed to confirm the causative role of a microorganisms in human disease (see Chapter 1).3 Yet even Koch himself recognized from the beginning that these guidelines were not relevant for all pathogens. Koch famously observed that leprosy was likely caused by the mycobacterial-like bacillus (Mycobacterium leprae) that was readily identifiable on tissue stains of involved skin, yet he (or anyone else for the next century) was never able to grow the organism on artificial media. This violated his own third postulate; nonetheless, he was comfortable assigning a pathogenic role to these readily visible leprosy bacilli. A myriad of well-known human pathogens, including many viruses, rickettsia, spirochetes and other pathogens, do not fulfill all of Koch’s postulates and yet their etiological identities are well established. The fact that many opportunistic potential pathogens colonize people for prolonged periods without causing disease (e.g. Candida albicans, Staphylococcus aureus, etc.) was not appreciated in the early days of clinical microbiology. This violates Koch’s second postulate. Moreover, many human diseases caused by well-known microbial pathogens lack comparable animal models (e.g. rheumatic fever, or progressive multifocal leukoencephalopathy) and therefore do not meet all of Koch’s postulates. The value and limitations of Koch’s postulates are described in detail in Chapter 1 in this text.
Alternative Views of Microbial Causation of Human Disease in the Genomic Era Periodontitis is among the most prevalent infectious diseases, afflicting 30–70% of adults worldwide.7 This oral infection has been extensively studied by both culture-dependent and culture-independent methods.8
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A consistent pattern of microbial pathogens has now emerged as the cause of periodontitis. The genomic evidence strongly supports the communal cause of periodontal disease. Biofilms consisting of Streptococcus mutans and other oral streptococci residing in a mono- or disaccharide-rich environment in the oral cavity express exotoxins that gradually induce dental caries over time. A synergistic combination of bacterial pathogens living within the biofilm lining the gingival crevice then gives rise to periodontal disease. A combination of Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans gradually leads to gingivitis, bone resorption and the full expression of periodontitis. Later disease progression often involves additional noncultivable bacterial strains and a methanogenic Archaea organism (the first example of a member of the Archaea kingdom associated with a human disease).1,2 Important human illnesses are the result of long-term sequelae of remote infections (the ‘hit and run’ hypothesis) that occur months or even years after the infection has cleared. Examples include Guillain– Barré syndrome following enteric infection with specific serotypes of Campylobacter spp.; Sydenham’s chorea as a late manifestation of the non-suppurative complications of S. pyogenes pharyngitis; or postinfectious reactive arthritis (formerly known as Reiter’s syndrome) following enteric infections. The pathogen is usually no longer cultivable from these patients when the disease is manifest and yet their immunologic footprint clearly identifies the etiologic organism in each case.1,6 A large number of immune-mediated, inflammatory and neoplastic diseases may have, as their proximate cause, a specific pathogen or group of micro-organisms as an inducer or essential propagator of human illness. Exposure of the susceptible host with the appropriate genotype, and the fortuitous concurrence of the necessary set of environmental and epidemiologic circumstances conspire together to initiate and maintain an aberrant host response leading to overt clinical disease. Sarcoidosis might be an example of a disease induced by environmental and generally nonpathogenic bacterial organisms in some genetically susceptible individuals.9–12 A specific genetic locus has been recently identified that appears to be linked with sarcoidosis.13 Excessive and aberrant granulomatous reactions to common microorganisms such as Propionibacterium acnes14–16 or tuberculous or environmental, non-tuberculous, mycobacterial antigens17,18 might induce sarcoidosis in these susceptible patients. Similarly, genetic susceptibility to inflammatory bowel disease is well described in association with NOD2 polymorphisms and other mutations in pattern recognition receptors (Toll-like receptor 4) resulting in defective clearance of enteric bacteria and microbial antigens and mediators along the mucosal epithelium of the small and large bowel.19–24 This genetic predisposition to inflammatory bowel disease, in concert with alterations in the normal microenvironment and enteric microbial flora, is currently one of the favored hypotheses to explain the pathogenesis of Crohn’s disease.25–27 Endemic Burkitt’s lymphoma in children in sub-Saharan Africa provides another unfortunate example of a confluence of permissive environmental conditions and collaboration between two widely disparate pathogens to cause neoplasia. Burkitt’s lymphoma is common in African children living in holo-endemic regions where mosquitoborne falciparum malaria exists. Neoplastic transformation to lymphoma is generally attributed to an aberrant host response to Epstein–Barr virus (EBV) infection. Recent evidence indicates that Plasmodium falciparum, via its major erythrocyte protein PfEMP-1, induces general immunosuppression and profound B-cell proliferation that activates productive infection with EBV. High-level replication of EBV promotes the translocation and activation of the potent oncogene c-myc resulting in malignant transformation to Burkitt’s lymphoma.28,29 The potential role of other viral, bacterial and parasitic infectious diseases in the pathogenesis of other neoplastic diseases is surveyed in Table 69-1.28–31 There are a large number of clinical settings in which a specific micro-organism or group of micro-organisms might be necessary and sufficient to cause a variety of human illnesses, yet go unnoticed when
TABLE 69-1
Microbial Disease Associations with Neoplastic Diseases Level of Evidence of Causation*
Possible Pathogen
Neoplasia
Helicobacter pylori
Gastric adenocarcinoma Gastric lymphoma
+++ ++
Salmonella typhi
Biliary carcinoma in carriers
+++
EBV
Burkitt’s lymphoma (with Plasmodium falciparum) Hodgkin’s disease Nasopharyngeal carcinoma Primary CNS lymphoma Immunoblastic lymphoma
+++
Hepatitis B
Hepatoma
+++
Hepatitis C
Hepatoma
+++
Schistosoma haematobium
Bladder carcinoma
++
Clonorchis sinensis
Cholangiocarcinoma
++
HPV-16/18
Cervical carcinoma Colorectal cancer
+++ +
Helicobacter bilis
Biliary tract cancer
+
HTLV-1
Acute T-cell leukemia/lymphoma Invasive cervical carcinoma Small cell carcinoma of the lung
HERV-K10
Testicular cancer
Kaposi’s sarcomaassociated herpesvirus (HHV-8)
Kaposi’s sarcoma Primary effusion lymphoma
++ ++ +++ ++
+++ + + + +++ ++
*Level of evidence: epidemiological link only +; some evidence of causation ++; clear evidence of causation +++. CNS, central nervous system; EBV, Epstein–Barr virus; HERV, human endogenous retrovirus; HH8-V, human herpesvirus 8; HPV, human papilloma virus; HTLV, human T-cell leukemia/lymphoma virus.
relying upon standard diagnostic techniques.1–5 A number of epidemiological features and histopathologic findings suggest a microbial cause for a variety of idiopathic diseases. Such diseases as sarcoidosis,9–11 Kawasaki disease,32 multiple sclerosis,33 type 1 diabetes mellitus,34 Kikuchi–Fujimoto disease35–37 and numerous others suggest an underlying infectious cause. Caution needs to be exercised before it can be unequivocally concluded that the implicated organism is in fact the true etiologic agent.3,6 Certain organisms could be highly associated with a specific disease entity, yet simply occupy a unique ecologic niche created by the disease itself. Streptococcus gallolyticus is highly associated with neoplastic lesions in the colonic mucosa, yet there is little evidence to suggest that this organism causes colon tumors. The organism takes the opportunity to colonize the unique microenvironment adjacent to the neoplastic tissue. A survey of potential mechanisms by which the microbial cause of disease could go unrecognized using conventional diagnostic methods is provided in Table 69-2.38–41
The Hygiene Hypothesis and the Pathogenesis of Inflammatory Diseases It should also be acknowledged that, paradoxically, heavy exposure to some elements of the microbiota, particularly at a young age, may actually protect against a number of inflammatory and allergic diseases later in life. The incidence of asthma, Crohn’s disease, diabetes and multiple sclerosis has progressively increased over the last half century in higher-income nations as vaccine-preventable, and food- and
Chapter 69 The Potential Role of Infectious Agents in Diseases of Unknown Etiology
TABLE 69-2
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Mechanisms by which Causative Microbial Pathogens Might Escape Detection
Mechanism
Examples
Comments
‘Hit and run hypothesis’
Streptococcus pyogenes – chorea minor in rheumatic fever Measles – subacute sclerosing panencephalitis
Disease manifestations induced by infection but separated from body site and in time by months, years or decades
Difficult to culture or noncultivable
Tropheryma whippleii – Whipple’s disease Bartonella henselae – cat scratch disease
Genomic studies indicate enormous numbers of microbial genomes that have yet to be successfully cultured
Commensal organisms that rarely cause disease in susceptible hosts
Sacrocystis spp. – enteritis or myositis Non-tuberculous mycobacteria – respiratory infections
Difficult or impossible to prove Koch’s postulates
Disease occurs with microbial communities
Periodontitis Irritable bowel syndrome Obesity? Bronchiectasis Bacterial vaginosis
Synergistic combinations of micro-organisms cause disease
Micro-organism induces immunologic or neoplastic alterations
KSHV (HHV-8) and Kaposi’s sarcoma Helicobacter pylori and gastric carcinoma
Microbial pathogen may serve as promoter or essential inducer
HERVs
HERV-E and systemic lupus erythematosis
Widely prevalent in human genome – possible inducer or propagator for chronic diseases
Different micro-organisms trigger illness only in genetically susceptible patients
Possibly sarcoidosis Kawasaki disease Multiple sclerosis, etc.
A variety of possible microbial pathogens may induce disease at critical time intervals in susceptible individuals
HERV, human endogenous retrovirus; HHV-8, human herpesvirus 8; KSHV, Kaposi’s sarcoma-associated herpesvirus.
TABLE 69-3
Koch’s Postulates Revisited: Microbial Causation in the Genomic Era
Modified Hill and Evan’s Criteria
Genomic Criteria
Prevalence of disease higher in patients exposed to suspect pathogen(s) than those not exposed
Nucleic acid sequence of suspect pathogen should be present in diseased tissues
Incidence of disease should be higher in patients exposed to suspect pathogen(s) than controls in prospective studies
No or few copies of nucleic acid sequences of suspect pathogen should be found in the absence of disease and in normal tissue
The disease should be temporally linked to exposure to suspect pathogen(s)
If suspect pathogen sequences predate onset of disease, the copy number of sequences should increase with onset of clinical illness (temporal relationship)
A spectrum of disease should be found after exposure from mild to severe disease (dose–response)
Copy number of suspect pathogen sequences should correlate to disease severity (gene copy – host–response relationship)
A measurable host response should occur following exposure to suspect pathogen(s)
The suspect pathogen sequences should localize by in situ hybridization to affected tissues
Experimental reproduction of disease should occur in models or human volunteers
Suspect pathogen sequences should be reproducibly found in patients with similar illnesses and in animal models (if available)
Prevention of transmission of suspect pathogen should decrease incidence of disease and eradication of pathogen should decrease the disease
Resolution of clinical illness should be accompanied by reduction or elimination of suspect pathogen sequences
The whole process should make biologic sense and fit the epidemiology of disease
The suspect pathogen determined by nucleic acid homology searches should fit the biology and pathology of genetically related micro-organisms
water-borne enteric infections have dramatically decreased.42 These inflammatory diseases are much less common in low- and middleincome countries where the incidence of childhood infection remains high. The ‘hygiene hypothesis’ suggests that improved hygiene fails to prime innate immune responses to common microbial antigens, particularly chronic geo-helminth infections, in early life. This impairs mucosal immune maturation, promotes a TH2-type cytokine response and facilitates inappropriate cellular tolerance to many common microbial antigens. The end-result is excessive and disordered inflammation to many of these environmental antigens later in childhood and adulthood with resultant increased risk of inflammatory diseases. The hygiene hypothesis seems plausible yet still lacks extensive, experimental evidence in support of this appealing notion.42-45
Culture-Independent Techniques to Detect Novel Pathogens The evolving definition of what constitutes a human pathogen has moved from a formal requirement to fulfill all of Koch’s postulates to a genomic expression of Descartes’ assertion, ‘I clone, therefore I am’ (Table 69-3). This is best evidenced by the remarkable discovery of hepatitis C as the most common cause of post-transfusion hepatitis by Choo et al. in 1989.46 After decades of concerted, yet futile, efforts to isolate the implicated virus using standard virologic techniques, they attempted to clone the genome of the unknown agent directly from the serum of a chimpanzee experimentally infected and known to contain high titers of infectious agent.
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Since it was not known if it was an RNA or DNA virus, they treated the serum with reverse transcriptase and generated a large number of sequences of amplified nucleic acids to create a comprehensive phagebased cDNA library. These clones were tested against the serum of a patient with chronic, post-transfusion hepatitis in hopes the serum would contain antibodies that recognize at least some of the expressed viral peptides. After screening nearly one million clones, the reactive peptide sequence was identified. They then showed that this was an RNAse- but not DNAse-sensitive genome and, by overlapping hybridization experiments, they confirmed that this was a positive-stranded RNA flavivirus of approximately 10 000 base pairs. The mystery of post-transfusion hepatitis was finally solved by cloning, unencumbered by the need to fulfill Koch’s postulates.3,46-48 A similar cloning strategy has now been adapted from work done with genome searches for comparative microbial ecology, environmental biology and molecular phylogeny studies.2,3 Microbial DNA sequences are isolated and amplified by polymerase chain reaction (PCR) using broad range bacterial primers for essential target sequences, such as ribosomal RNA genes. These amplified, novel bacterial sequences are then cloned in a DNA library where comparative sequence analysis is performed with highly conserved sequences from known classes of micro-organisms. The genes for 16S RNA, other essential ribosomal RNA sequences, or highly conserved enzymes are scanned for sequence homologies throughout the Bacteria and Archaea kingdoms. Sequence homologies and genomic groupings can now be ex ploited to identify shared genetic space and evolutionary distance between essentially any unknown pathogen and all identifiable, yet noncultivable, genome-characterized micro-organisms that might cause human disease. Such unbiased genome searches have been instrumental to the work of Relman, Fredricks and others3 in discovering the etiology of longstanding diseases of known microbial cause, yet not cultivable organisms such as the Whipple’s disease bacillus (Tropheryma whippleii).49,50 A genome search of the organism revealed mutations in metabolic genes and the nutritional requirements needed to successfully grow the organism on artificial media for the first time.51 Newly recognized, emerging pathogens such as the causative agents of bacillary angiomatosis (Bartonella henselae) and human monocytic ehrlichiosis (Ehrlichia chaffeensis) were first identified using a similar genomic search strategy3 (Figure 69-1). Current sequencing machinery now permits the simultaneous sequencing of short unique nucleotides from small samples of DNA without the need for preparatory cloning3 with rapid genome screens with known sequences of viruses, rickettsia and numerous other difficult-to-culture pathogens. DNA subtraction is another technique that can be used to detect and define novel microbial pathogens. This methodology was successfully employed to link a previously unknown herpes-like virus (HHV8), now known as Kaposi’s sarcoma-associated herpesvirus (KSHV), as the cause of HIV-related Kaposi’s sarcoma.2,3 The DNA sequences derived from diseased tissues and a similar set of DNA sequences from normal tissues are subjected to subtractive hybridization and PCR amplification to enrich the DNA complement of DNA sequences found in the diseased sample only. Unique sequences are then analyzed to determine if any sequence homologies exist with known pathogens. As the known genomic universe of micro-organisms continues to expand, it is likely that this and related culture-independent methods will reveal new pathogens associated with human illness.52
THE ROLE OF HUMAN ENDOGENOUS RETROVIRUSES (HERVS) IN HEALTH AND DISEASE The human genome is replete with endogenous retroviruses (HERVs, also known as retrotransposons) that have entered the human germline at various times in the evolutionary past and now occupy 8.3% of the genome.53 They maintain the basic structure of retroviruses with long terminal repeats (LTR) flanking the open reading frames for polymerase (pol), including reverse transcriptase, envelope (env) and
A genomic strategy to search for novel microbial pathogens as a cause of human disease
Isolate DNA and RNA sequences from clinical material from diseased tissues Suspect pathogen
RNA
DNA
+RT cDNA
PCR
DNA
Shotgun clone DNA amplicons into plasmid vector DNA to generate a DNA library
Sequence novel DNA segments
Screen clones with specific anti-sera if available (e.g. HCV)
Search for alignment for homology with known RNA and DNA genomes or highly conserved sequences (e.g. 16S rRNA)
Confirm
Develop oligonucleotide probes or primers and search involved tissues for in situ hybridization or in situ PCR
Attempt to culture suspect pathogen using techniques for related micro-organisms
Figure 69-1 A genomic strategy to search for novel microbial pathogens as a cause of human disease. RT, reverse transcriptase; cDNA, complementary DNA; PCR, polymerase chain reaction; HCV, hepatitis C virus.
core matrix (gag) genes along with various regulatory genes. They have accumulated loss of function point mutations in structural and regulatory genes rendering them incapable of exogenous viral production. It is possible to ‘resurrect’ some recently inherited HERVs from the human genome but this is unlikely to occur spontaneously.54 However, these endogenous retroviruses are not genetically dormant. Some HERVs have physiologic roles, including syncytiotrophoblast formation in placental development and intrinsic resistance to exogenous retrovirus infection.55 HERVs also have the potential to contribute to pathologic reactions in their hosts as well as by a number of mechanisms.53 The LTR regions are transcriptionally active and serum antibodies are detectable to env and gag proteins from a number of HERVs, indicating that these structural genes are transcribed and expressed. Transposition of HERVs can inactivate cellular genes at other loci in the human genome. Moreover, the LTRs of HERVs can have polar effects in cis and promote transcriptional activation of adjacent cellular genes. Lastly, transcriptional activators are found within HERVs that might have the capacity to activate cellular genes in trans. HERVs have been implicated in induction of malignant transformation by activating oncogenes or inactivating apoptosis-inducing genes. HERVs might contribute to immune-mediated diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE) by the generation of HERV antigens that crossreact with endogenous antigens and break tolerance to normal
Chapter 69 The Potential Role of Infectious Agents in Diseases of Unknown Etiology
autoantigens. The gag p30 of class I HERVs bear striking homology with the human 70K ribonucleoprotein U1 snRNP, a frequently recognized autoantigen in SLE.56 SLE might be induced in some patients by molecular mimicry when expressed HERV gag proteins induce antibodies that cross-react with epitopes found on the 70K RNP. HERVfacilitated epitope spreading and loss of Fas-mediated apoptosis of self reacting T-cells might further contribute to autoimmune disease progression. HERVs may collaborate with other viruses, such as EBV, to induce the synthesis of endogenous superantigen motifs encoded by HERVs.57 This process is hypothesized to contribute to the immune-mediated CNS pathology in MS. HERV-K18 has superantigenic activity in its envelope peptide; expression of this Env protein is transactivated by B cell clonal expansion by co-infection with EBV. This type of immune dysregulation with the CNS is implicated in the pathogenesis of white TABLE 69-4
629
matter lesions in MS.53,57,58 Finally, recent evidence suggests that the Env protein of HERV-W can act as a ligand for the pattern recognition receptors CD14 and TLR4.59 Activation of effector cells of the innate immune system via TLR4 with cytokine and chemokine generation might further contribute to injurious CNS inflammation in MS and other demyelinating disorders. A brief review of infectious diseases that have been associated with or cause inflammatory and vasculitic diseases is presented in Tables 69-4 and 69-5.60–67
Conclusions The number of potential pathogens that might cause disease in some susceptible human populations has expanded by an order of magnitude with the recognition of the array of noncultivable micro- organisms in our environment and even among our endogenous
Microbial Disease Associations with Inflammatory Diseases
Possible Pathogen
Disease
Level of Evidence of Causation*
Propionibacterium acnes
Sarcoidosis
+ (environmental mycobacteria also implicated)
HHV-6
Multiple sclerosis (relapsing-remitting type)
+/++ (HERV-W also implicated in association with EBV or HHV-6)
Campylobacter jejuni
Cross reacting anti-ganglioside antibodies and Guillain-Barré syndrome
+++
Mycobacterium paratuberculosis
Inflammatory bowel disease
+ (high concentrations of enteric bacteria also implicated)
HERV-W
Psoriasis
+
HERV-K18
Type I diabetes
+ (enteroviruses also implicated)
Bacterial superantigens
Kawasaki disease
+
Yersinia enterocolitica
Kikuchi–Fujimoto disease
++ (KSHV also implicated)
KSHV
Multicentric Castleman’s disease
++
Mixed enteric aerobes and anaerobes
Necrotizing enterocolitis63
+
Enteric viruses
Onset of type 1 diabetes mellitus
+
Chlamydia spp. and enteric bacteria
Ankylosing spondylitis
+
*Levels of evidence: epidemiological link only +; some evidence of causation ++; clear evidence of causation +++. EBV, Epstein–Barr virus; HERV, human endogenous retrovirus; HHV, human herpesvirus; KSHV, Kaposi’s sarcoma-associated herpesvirus.
TABLE 69-5
Microbial Disease Associations – Vasculitis
Microbial Agent
Disease
Level of Evidence of Causation*
Hepatitis B
Polyarteritis nodosa, membrano-proliferative glomerulonephritis
++/+++
Hepatitis C
Mixed cryoglobulinemia
++/+++
Staphylococcus aureus
Granulomatosis with polyangiitis (formally Wegener’s granulomatosis) Churg–Strauss eosinophilic vasculitis
++ +
EBV and/or CMV
Rheumatoid arthritis
++ (HERVs also implicated)
CMV
Atherosclerosis
+ (Chlamydia pneumoniae also implicated)
Human endogenous retrovirus-E
Systemic lupus erythematosis
+ (EBV also implicated)
Parainfluenza virus
Giant cell arteritis
+ (parvovirus B19 also implicated)
Bacterial lipopolysaccharide
Antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis
+
Helicobacter pylori
Sjögren’s syndrome, atherosclerosis
+
Propionibacterium acnes
SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis)
++
*Levels of evidence: epidemiological link only +; some evidence of causation ++; clear evidence of causation +++ CMV, cytomegalovirus; EBV, Epstein–Barr virus; HERV, human endogenous retrovirus.
630
SECTION 3 Special Problems in Infectious Disease Practice: Fever
microbiota. It is possible that at least some human diseases of unknown cause will eventually be attributable to noncultured organisms. Synergistic combinations of viral, bacterial and even parasitic infections are now known to cause specific clinical disease entities, and it is likely that other disease entities are caused by communal groups of microorganisms. Aberrant host responses to common micro-organisms
might explain many inflammatory and perhaps even some neoplastic diseases. The role of human endogenous retroviruses in human disease, alone or in combination with other potential pathogens, remains a fertile area of research in the coming years. References available online at expertconsult.com.
KEY REFERENCES Fredricks D.N., Relman D.A.: Sequence-based identifica tion of microbial pathogens: a reconsideration of Koch’s postulates. Clin Microbiol Rev 1996; 9: 18-33. O’Connor S.M., Taylor C.E., Hughes J.M.: Emerging infectious determinants of chronic diseases. Emerg Infect Dis 2006; 12(7):1051-1057.
Pendergraft W.F., Preston G.A., Shah R.R., et al.: Autoimmunity is triggered by cPR-3 (105-201), a protein complementary to human autoantigen proteinase-3. Nat Med 2004; 10(1):72-79. Ridaura V.K., Faith J.J., Rey F.E., et al.: Gut Microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013; 314:1079.
Weng L., Rubin E.W., Bristow J.: Application of sequencebased methods in human microbial ecology. Genome Res 2006; 16:316-322.
Chapter 69 The Potential Role of Infectious Agents in Diseases of Unknown Etiology 630.e1
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