Increasing numbers of immunocompromised patients Ever-evolving life-sustaining technologies Broadening chemotherapeutic protocols Immuno-therapeutic protocols Transplanatation medicine Expanding antimicrobials and resistance patterns

Responsibilities of the Anatomic Pathologist:
The goals and approaches used by the microbiologist, cytopathologist and surgical pathologist are more similar than many realize. Computer technologies further enhance our ability to function as a team, on behalf of the patient. Overlap in the clinical workup of specimens strengthens the diagnostic certainty and potential for reaching the ultimate goal – an infectious disease diagnosis with a confirmed etiologic agent.

The charge for the anatomic pathologist in today's era of infectious disease pathology is three-fold. We are expected to:

1. Optimize opportunities to intervene and guide in collection of appropriate cultures based upon rapid evaluation techniques within the surgical pathology and cytology laboratories;
   
   
2. Maximize availability of limited tissue samples for multiple diagnostic modalities, including molecular techniques, by using a systematic approach to the laboratory work-up; and
   
   
3. Formulate final anatomic diagnoses that correlate clinical history with histologic findings, special stains, immunohistochemistry, follow-up cultures and molecular studies.

Clinical History
The clinical history is of paramount importance when evaluating patient specimens. It goes without saying that straightforward cases such as respiratory infections with common agents such as Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas and Staphylococcus aureus, are easily diagnosed by blood or respiratory cultures and almost never make it to the bronchoscopy and/or OR suite. The pathologists are often left to deal with the 'uncultureable' undiagnosed medical 'mysteries.' Known risk factors, zoonotic exposures, tick/insect exposures, work exposures, age and immune status are some of the critical elements required.

The complexity increases tremendously when we consider infections of the immunocompromised host. Pneumocystis jirovecii (carinii) in classic form with abundant protein-rich alveolar casts and abundant organisms is not usually a diagnostic challenge. However, given the unusual pneumocystis presentation: hyaline membranes rich type; granulomatous and interstitial pauci-reactive, the pathologist can be caught off-guard unless pertinent history is provided. The patient with suppressed immune status may produce no-granulomas or poorly formed granulomas in the setting of overwhelming Mycobacterium tuberculosis and systemic fungal infections. One must also consider a much broader differential in the immunocompromised host, giving consideration for agents such as Rhodococcus equi in the setting of a patient presenting with cavitary lung lesions, but negative for mycobacteria. In the transplant patient population, consideration must be given to viral induced lymphoproliferations such as Epstein-Barr Virus infection.

Gross Examination and Rapid Assessment techniques
A careful gross examination with preparation of frozen sections, direct smears and/or imprint slides, as appropriate, is critical. Touch imprints, direct smears, and tissue scrape or squash preps can all be used for preparation of either fixed or air-dried slides. These can be used for rapid special stains or saved for use if needed after routine sections are reviewed. A rapid evaluation will often clue the pathologist when there is a need for sequestering fresh tissues for flow cytometry or frozen for possible molecular or immunologic studies. There is no turning-back once the specimen is plunged into fixatives.

Microscopic Examination (Routine and Special Stains)
Never underestimate the importance of the routine Hematoxylin-Eosin stain. Many viral infections and most parasitic infections can be quickly and easily recognized using this age-old standard. The H&E provides a wealth of information about the immune response pattern and guidance allowing an informed, systematic approach to special stain orders.

A variety of histochemical stains can be used for evaluation of infectious agents. None are specific, but in combination with the morphology of the organism, the host response, and the clinical history a presumptive etiologic diagnosis may often be rendered with a high level of certainty. The Gram stain remains the most useful rapid diagnostic technique in the microbiology laboratory. Similarly, the hematoxylin and eosin (H&E) stain remains the mainstay of the histology laboratory and surgical pathologist. For the cytologist the Wright stain and Papaniculaou stains are the mainstay.

Numerous immunoperoxidase stains have now been developed for both common and rare pathogens. Commercial reference laboratories can often assist you, if your test volumes do not permit you to keep these antibodies in-house and ready for use. For the rarest of stains, the Center for Disease Control and Prevention in Atlanta, GA is an excellent resource. Contacting the pathology branch for guidance is recommended.

Direct Immunologic Detection
Direct immunologic detection of infectious agents in smears and tissue provides increased specificity and sensitivity as well as an additional level of certainty for the definitive identification of infectious disease agents. This must be done in combination with the clinical history and knowledge of the appropriate inflammatory reaction in the tissues under investigation. Techniques used for this purpose include direct or indirect immunofluorescence assays and enzyme linked immunochemistries. The marker for these immunochemistry methods may be horseradish peroxidase, alkaline phosphatase, or biotin-avidin compounds. Commercial reagents are available for immunologic detection of many viruses, such as HIV, herpes simplex virus, cytomegalovirus, hepatitis C and adenovirus; for bacteria, such as Legionella spp. and T. pallidum; and for parasites, such as Toxoplasma gondii.

Culture and Serologic Correlation
It is important to correlate the surgical pathology and cytopathology findings with both culture and serologic findings. In the current age of computer connectivity, this is rarely problematic. Clinicians can often be guided in follow-up cultures or serologic testing that may help confirm the anatomic pathology findings.

Molecular Diagnostics
Evolving technology has driven pathologists to a new level of service in the realm of infectious disease pathology. Culture and sensitivity techniques continue to be refined and molecular applications provide a rapid method for determining organism load, virulence factor traits and invaluable genomic information. Although culture still remains the gold standard, molecular diagnostics allows us to confirm the presence of the "unculturable" agents and to tremendously improve the turn-around-time for those organisms requiring prolonged incubation times and tedious confirmation steps. In addition, when the histologic features suggest an unsuspected infectious process molecular techniques can confirm special stain findings in the absence of available cultures.

When considering any new test methodology or technology it is always helpful to step back and consider the basics of laboratory medicine. What are the strengths and limitations of a new technology – all tests even molecular amplification methods have limitations. Other considerations include cost, expertise and added value. The National Committee for Clinical Laboratory Standards (NCCLS) has developed a set of recommendations to consider specifically when evaluating new molecular technology. Although the health care industry looks to the FDA as the final sanctioning body for new testing methodologies, the FDA simply has not been able to keep up with the vastly expanding numbers of new molecular tests and methodologies. We must scrutinize each of these new applications carefully prior to adopting them for our patient populations. We must compare and contrast sensitivity, specificity, precision, accuracy, method-specific limitations and cost for every new test we consider utilizing. This puts a tremendous burden on the pathologist who is expected to be the expert for all laboratory testing methodologies.

The Immune Response Pattern
Attention to tissue responses may provide the initial clues in the search for causality. Tissue reactions allow pathologists to categorize injury patterns and then group potential suspect infectious agents. The search is then narrowed by application of a vast array of identification methods and techniques currently available

Although there are thousands of potential infectious agents we may encounter, there are a very limited number of immune responses to those agents. A particular type of pathogen usually elicites a predictable response based on the mediators and potentiators provided by the organism and the composition of the cytokine response to the invading pathogen. The local tissue injury occurring with the initial contact may also cause further injury and potentiation of the inflammatory response. The lipopolysaccharide component of the gram negative bacilli external membrane stimulates an intense release of IL I, tumor necrosis factor, IL 6 and IL 8 leading to a predominately neutrophilic response. On the other hand invasion of an obligate intracellular pathogen, such as a virus or a rickettsial organism stimulates the release of IL 1, IL 2 and interferon leading to a mononuclear cell mediated response. Because there are predictable responses with specific categories of infectious agents, we can make certain generalizations that provide a framework that narrows and focuses our search when dealing with an unknown infectious disease pathogen. It comes as no surprise that there are exceptions when using this approach. Nonetheless, we can begin an immune response guided systematic approach to identification of the unknown pathogen. Seven major types of histologic responses can be delineated as follows:

Exudative acute inflammatory reaction Necrotizing inflammatory reaction Granulomatous inflammatory reaction Mononuclear, predominately foamy macrophage reaction Lymphoplasmacytic reaction Cytopathic/direct injury reaction Paucicellular or 'absent' reaction

It should also be noted that some infections are not associated with a recognizable cellular reaction in the affected tissue. The most common explanation for serious infections without an inflammatory response is that the pathogenic mechanism of the infection is production of a toxin that exerts its effects biochemically. Two examples are cholera, which is a disease of intestinal fluid secretion, and botulism, which is a disorder of neuromuscular transmission. A second completely diverse explanation is that the immune system of the infected host is either severely suppressed or they are anergic. Both of these settings could result in the paucicellular or "absent" response pattern.

Tissue Reaction Patterns and Potential Etiologic Agents for Consideration

Tissue Reaction Pattern	Injury Mechanism	Potential Etiologic Agents	Methods for Confirmation
Exudative Inflammatory Rxn	NF-kBpathway; IL-1; TNF; IL6;IL8; IFN-γ	Pyogenic bacteria; rapid growing organisms; legionella	Culture; H&E; Tissue gram; Silver ppt stain; PAS
Necrotizing Inflammatory Rxn	Toxins; Leukocidins	Clostridium; legionella; pseudomonas; pyogenic bacteria; E. histolytica.	Culture; H&E; Tissue gram; trichrome; PAS
Granulomatous Inflammatory Rxn	TH-1;IL1, IL2; TNF; IL6; IL8; IL10.	Mycobacteria; fungi; rare gram negatives; helminth larvae/eggs.	Culture; H&E; Silver; AFB; gram; PAS; trichrome; molecular
Mononuclear, predominately foamy macrophage Rxn	IL1; IL2; IL6; IL8; MCP; MIP;IL12, IL15	Fungal, intracellular and intravascular agents; Rhodococcus; PCP	Culture; H&E; gram; geimsa; AFB, GMS; molecular; serology.
Interstitial,predominately lymphocytic Rxn	IL1; IL2; IL6; IL8; MCP; MIP;IL12, IL15	Fungal, intracellular organisms bacteria; protozoa	Culture; H&E; gram; giemsa; AFB, GMS; PAS; molecular; serology.
Cytopathic/Direct Injury Rxn	Apoptosis; receptor entry; NK, CT cells	Viral agents; parasitic infections	Culture; H&E; immunohistology; molecular.
Paucity or "Absent" Rxn(must consider immune status of host!)	Low virulence and cellular mimicry	Latent viruses;Low virulence bacteria/mycobacteria; parasites.	Culture; H&E: gram; giemsa; AFB, GMS; PAS; molecular; serology.

Note: In cases demonstrating overlap features, first consideration should be given to the over-riding pattern.

Recent and potential future challenges:
The United States has been challenged by both nature and the threat of additional potential terrorist events occurring in our country. Pathologists and the laboratory are a vital link in our preparedness for a natural or terroristic biologic event. Some of the recent events include:

Anthrax mailings of 2001 Small pox threat West Nile Virus Avian Influenza Norovirus (Norwalk like) SARS

It goes without saying that all methods and diagnostic tools used by the pathologist in both the clinical and anatomic realm have their limitations in sensitivity and specificity. Even when an amplification molecular method is "negative" it cannot completely rule out the presence of an organism. Organisms don't always follow the rules, and although targeting the 'most likely pathogen' list based on immune response and tissue reaction pattern makes perfect sense, it doesn't always disclose the precise etiologic agent.

When the history of a patient is known and discussed by the pathologist and internist in advance the best outcome will occur. This will allow for advanced planning to include the most appropriate cultures, including bacterial, fungal, mycobacterial and comprehensive viral cultures; touch preparations for rapid stains, as clinically needed, and ordering of appropriate special stains at the time of tissue processing to improve final turn-around-time. In addition, consideration may be given to set tissue aside frozen for direct fluorescent stains and/or molecular studies. The need for additional studies such as flow cytometry can quickly be assessed via touch preparation, scrape preparation or frozen section.

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