This is an exciting time for research on pathogenesis and the immune response to infectious agents. The articles included in this issue illustrate the rapidly expanding knowledge regarding microbial virulence factors, the acute and chronic response to wound infections, and so on.
Recent discoveries on virulence mechanisms have resulted in new methodologies for rapid detection, vaccine development, and in-depth epidemiologic studies which include whole genome sequencing of related pathogens. In the clinical microbiology laboratory, rapid pathogen identification by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy has reduced the work load and obviated the need for time-consuming classical methodologies using a myriad of biochemical tests. However, these advances combined with computerized ordering and reporting of test results has clearly reduced critical interactions between physicians and clinical microbiologists. This has also resulted in greater reliance on standardized treatment algorithms while de-emphasizing the value of more basic diagnostic skills.
In contrast to modern times, early pioneers in the field relied heavily on simple tests, a careful history from the patient, and a thorough physical examination. Diagnoses were based on the appearance and evolution of local or systemic signs and symptoms including fever as well as careful microscopic examination of bodily fluids and evaluation of peripheral blood smears. How exciting it must have been to use these clinical features to establish a definitive diagnosis of malaria, trypanosomiasis, leptospirosis, and cholera to mention a few. Even with modern technological advances, the observations and relationships established by early practitioners have stood the test of time and, in this author's opinion, such clinical acumen is still crucial for a timely and correct diagnosis.
FEVER: HISTORICAL PERSPECTIVES AND MODERN CLINICAL SIGNIFICANCE
Before the advent of the microscope, the manifestation of fever was a vital clue to an accurate diagnosis. Practitioners were aware that the onset, degree, and pattern of fever were characteristic for specific types of infectious diseases. For example, the sudden onset and persistence of high temperatures for several days followed by sudden defervescence was characteristic of lobar pneumonia. This latter feature, termed ‘the crisis,’ was generally predictive of patient survival. In contrast, recurrent high fevers with periodic returns to normal were referred to as tertian (every 3 days), quartran (every 4 days), or malignant (persistent fever with spikes) and were characteristic of different types of malaria and were well recognized in endemic areas of plasmodium. Alternatively, cyclically increasing fever over 7–8 days followed by afebrile periods of 5–6 days was strongly associated with Hodgkin's disease (Pel–Ebstein fever) as well as tick-borne relapsing fever. Indeed, some of us remember the graphic temperature plots on bedside charts which have been replaced by tabular values on computers.
Early investigators and practitioners were unsure whether fever was beneficial or detrimental to the host. As the resolution of fever (crisis) was considered a good sign, some reasoned that fever must be deleterious. This notion was supported by the observation that patients (usually small children) with fever sometimes experienced febrile seizures. In contrast, other data demonstrated that modestly elevated temperature (38.5–40°C) increased leukocyte chemotaxis and phagocytosis, and improved macrophage and T-lymphocyte function in some cases. Temperatures more than 43°C were associated with decreased cellular function and denaturation of enzymes. The benefits and detrimental consequences of fever were elegantly reviewed by Mackowiak . More recently, Evans and colleagues  have reviewed the integrated physiological and neuronal circuits by which fever thermoregulates the immune system.
Because of fever's yin-yang characteristics, pharmacologic efforts to reduce fever have had differing consequences. Aspirin and acetaminophen have been shown to reduce fever but also to prolong viral shedding of rhinovirus and similarly prolong the time for crusting of lesions in children with chicken pox. Other reports have demonstrated a positive correlation between maximum temperature on the day of bacteremia and survival in patients with polymicrobial sepsis and those with spontaneous bacterial peritonitis. In patients with streptococcal pharyngitis, use of nonsteroidal anti-inflammatory drugs has been associated with increased complications such as peritonsillar abscess [3–5].
Despite the fact that fever patterns were remarkably and reliably pathognomonic, the genesis of fever remained enigmatic. Evil humors or ‘phlogistins’ produced by the various microbes were initially blamed, though thoughtful investigators such as Beeson  considered that fever resulted from the host response to infection. As proof of principle, he isolated a substance from human leukocytes, called ‘leukocyte endogenous mediator,’ that caused fever when administered to experimental animals . Others showed that there were two different fever-inducing molecules that could be purified either from the serum of febrile patients or from leukocytes stimulated with bacterial products such as lipopolysaccharide. Dinarello  proved that one of these molecules was interleukin 1 (IL-1). Beutler and Cerami  discovered a second pyrogen, called tumor necrosis factor (or cachectin), that not only induced fever but also mediated septic shock as well as rapid weight loss in cancer patients (cachexia) .
With fever clearly defined as a host response to infection, the question remained as to how a relatively limited repertoire of immune mediators were generated by the myriad of bacterial, viral, and mycobacterial pathogens that confront the human host. Ultimately, this question was answered by the discovery of Toll-like receptors on macrophages and other antigen-presenting cells that broadly recognized the various classes of pathogens (Gram positive, Gram negative, viral, and so on) via distinct microbial patterns . So significant was this discovery to modern immunology that the 2011 Nobel Prize in Physiology or Medicine was shared by Jules Hoffmann, Bruce Beutler, and Ralph Steinman who defined the mechanisms of pathogen pattern recognition by antigen-presenting cells.
Thus, the cause and consequences of fever as part of the innate host response was discovered precisely because basic science explanations were sought for important and long-standing clinical observations. Over the last 25 years, numerous other host-derived cytokines, interleukins, and lipid mediators have been discovered and have led to novel therapies designed to modulate the host response in patients with infection or immunologic diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease.
MICROSCOPY IN THE CLINICAL MICROBIOLOGY LABORATORY: ITS ROLE AND IMPACT ON MICROBIAL GENETICS AND IMMUNOTHERAPY
Basic science investigations into the pathogenesis of pneumococcal lobar pneumonia had a profound influence on clinical medicine. Prior to the advent of antibiotics, interest focused on the polysaccharide capsule – a recognized virulence mechanism for the organism. In particular, early clinical microbiology observations revealed that highly encapsulated smooth (S) Type III strains were more virulent than unencapsulated rough (R) Type II pneumococci. Work by Griffith  showed that an unknown acellular ‘transformative principle’ from a Type III culture supernatant could convert an unencapsulated Type II strain into a highly virulent Type III strain. In their seminal studies, Avery et al. unequivocally demonstrated that this principal substance was in fact bacterial DNA. These groundbreaking discoveries both shifted the prevailing paradigm in which proteins were believed to be the carriers of genetic information and solidified for the first time the ‘one gene, one protein (enzyme)’ concept. It also laid opened the door to all of microbial genetics.
Even before these studies, serotyping of pneumococcal strains was routinely practiced in clinical microbiology laboratories using serum from horses individually immunized with specific polysaccharide capsules from the multiple different strains of pneumococci. Type-specific protection against lethal pneumococcal challenge was afforded by passive immunization of mice with such sera. Investigators further demonstrated that such sera also enhanced phagocytosis of pneumococci with capsules comprised of only the specific polysaccharide used as an immunogen. Type-specific antibody recognition of pneumococcal strains was also demonstrated by microscopy in which the antibody–capsule interaction creates the appearance of capsular swelling (Quellung reaction). In New York and Boston City hospitals in the 1930s, pneumococci isolated from patients with lobar pneumonia or bacteremia was tested against banked horse sera using the Quellung reaction. When a positive horse serum was identified, said serum was administered to the patient. Patient survival was increased and significantly shortened hospital stays were realized. However, in private practice settings, misinterpretations of the Quellung reaction via microscopy led to poorer outcomes. Later, rabbits became the source of the anticapsular therapeutic.
Though therapy was effective, most patients developed serum sickness and when penicillin became available to the civilian population in the mid-1940s, this passive immunization strategy was abandoned . Nonetheless, this early work formed the basis for the current active vaccination products and the development of the rapid pneumococcal detection test.
MICROSCOPY IN THE CLINICAL LABORATORY: IMPACT ON EMERGING ANTIBIOTIC RESISTANCE
Microscopic interpretation of the Gram stain from sputum, liver abscess, bronchoalveolar lavage, urine, joint fluid aspirates, cerebrospinal fluid, or soft tissue samples should direct initial antibiotic coverage and each discovery should reduce excessive antibiotic use. Those microbiologists and practitioners that do not interpret Gram stains of patient specimens contribute to excessive antibiotic treatment as well as they rely solely on guideline-recommended protocols for empiric antibiotic therapy. In general, such guidelines recommend very broad-spectrum antibiotics that go far beyond that is necessary. In fact, a critical component of antibiotic stewardship is to make an early diagnosis and narrow the spectrum of antibiotic treatment. Careful and knowledgeable interpretation of Gram-stained material remains critical to early and accurate diagnosis. As rapid diagnostic procedures become the norm, particularly those amplifying DNA or RNA, microscopic evaluation of biological specimens remains vitally necessary as these techniques are highly sensitive and false-positives can arise from colonization as well as active infection. If a definitive diagnosis cannot be made, then broad-spectrum antibiotics will be used and generally for a prolonged period of time. This has ramifications in terms of development of multidrug-resistant pathogens and untoward complications such as Candida superinfections and certainly Clostridium difficile infections.
MICROSCOPY IN THE CLINICAL LABORATORY: IMPACT AND PITFALLS OF WHOLE BLOOD EVALUATION
Careful microscopic evaluations of blood smears provide unique opportunities to establish a better differential diagnosis or even a very specific one. Recent directives by clinical pathologists dictate that a manual differential count will be performed only if the white blood cell (WBC) count is less than 5 or more than 10 000 WBC/μl. This is problematic for multiple reasons. First, a patient with bone fide septic shock, or more specifically a patient with pneumococcal pneumonia, having a normal (or lower-end of normal) WBC is at increased risk for mortality. This is especially true if the patient is afebrile. In contrast, patients with pneumococcal pneumonia with elevated WBCs do better. Patients with group A streptococcal or staphylococcal toxic shock syndrome may also have a WBC in the normal to slightly elevated range, yet their peripheral blood smear shows a dramatic increase in bands, metamyelocytes, myelocytes, or even pro-myelocytes. This is a great clinical clue to the correct diagnosis.
In patients with clostridial infections including C. difficile, C. sordellii, or C. novyii, the WBC may be in the 15 000–250 000 WBC/μl range. In these infections, mortality is directly related to the degree of leukocytosis. These patients also have a marked left shift with increased bands, metamyelocytes, myelocytes, giant platelets, and nucleated red blood cells in the peripheral smear. This is obviously a danger sign from the host and should be an important component in making antibiotic recommendations or as rationale for emergent surgical evaluation for toxic megacolon (C. difficile) or gas gangrene (C. sordellii and C. novyii) even before a definitive diagnosis can be made.
For infectious disease and tropical medicine specialists, evaluation of the blood smear is also important to establish a diagnosis of leptospirosis, trypanosomiasis, and even malaria. Yet if the WBC is in the 5–10 000 WBC/μl range, microscopic examination of blood smears is not routinely performed. Practitioners should evaluate the blood smears in all patients with appropriate geographical exposures irrespective of a ‘normal’ WBC count.
PATHOGENESIS AND HOST RESPONSE IN THE HIV AND HEPATITIS ERA
Understanding the pathogenesis of HIV/AIDS is a prime example of how basic science research provided practitioners with extremely valuable tools to evaluate simultaneously both the status of the pathogen and the status of the human host. In the early years, those of us that managed patients with HIV truly had our hands full without much help. Technologies to enumerate viral loads and CD4 counts were not available and azidothymidine (AZT) was the only available treatment. Many patients succumbed to the usual secondary infections including cryptococcal meningitis, toxoplasma meningitis, disseminated Mycobacterium intracellulare infections, chronic wasting syndrome, and so on. Those that survived with AZT treatment were destined to have multidrug-resistant HIV infection.
Understanding the intricacies of the viral life cycle allowed development of protease inhibitors and newer agents that dramatically changed treatment and outcomes. Further this knowledge formed the foundation of newer diagnostic and monitoring technologies in which physicians can follow both the infection (viral load) and the host status (CD4 count). These advances have clearly improved management and outcomes in these patients. As a consequence, patients today that have had HIV for 30–40 years are now reaching normal longevity and succumb to general medical problems such as diabetes, hypertension, stroke, and myocardial infarction.
Similarly, basic science studies have yielded remarkable advances in hepatitis treatment. In the 1990s, hepatitis A and the newly diagnosed hepatitis B were well recognized. Other causes of viral hepatitis were called ‘non-A, non-B’ and modern technology determined the latter was in fact hepatitis C. Initial treatments were pegylated interferon and ribavirin, but their application was limited because of difficulties in identifying patients that did not use alcohol, did not have mental health issues related to depression because of the known side-effects of interferon, and who could adhere to 46–48 weeks treatment regimen. Very quickly PCR technology allowed determination of the ‘viral load’ of these patients under treatment and viral genotyping has improved targeted therapy. More recently, newer treatments without significant side-effects and with treatment durations of only 12–14 weeks have been employed with cure rates of 90%.
PAST LESSONS LEARNED AND THEIR CONTINUED RELEVANCE TO MODERN MEDICINE
In summary, in the last 50 years there has been great progress made by linking astute clinical observations to basic science investigations. We need to continue investigating these important relationships. Practitioners in developed nations should maintain proficiency in the older, simpler diagnostic tests and methodologies and consider them vital adjuncts to modern technologies.
On the global level, especially in which malaria is endemic, honed clinical acumen and observational skills are vital. In this setting, the presence of fever routinely prompts treatment for malaria because no methodologies, including basic microscopes, are even available to diagnose other infectious causes of fever. This crisis is discussed by Vogel  in which she makes it perfectly clear that such deficiencies delay recognition of emerging epidemics such as yellow fever, Lassa fever, and Ebola until they are out of control. Thus, basic laboratory and clinical skills remain critically important for global health and to prevent epidemics because of emerging pathogens that will, as history documents, occur particularly as climate change, war, famine, and explosive populating growth continue.
Financial support and sponsorship
Research Support: Support was provided by the Veterans Administration Basic Laboratory Research & Development Program, by a Center of Biomedical Research Excellence (COBRE) award from the National Institute of General Medical Sciences (NIGMS) under NIH Grant # P20GM109007 and by an Institutional Development Award (IDeA) from NIGMS under NIH Grant # P20GM103408.
Conflicts of interest
There are no conflicts of interest.
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