Introduction to infectious diseases
Everything about microscopic life is terribly upsetting … how can things so small be so important?
(Isaac Asimov—1920–1992)
An old enemy
Infectious diseases have had a huge impact on the human species. Throughout history, mighty armies have been humbled by microbiology. After the introduction of effective antibiotics during the Second World War, there was great optimism that the fight against infectious diseases had been won. In recent years, this hope has been dramatically dashed. Almost all new diseases are infections, and some of the twenty-first century’s most pressing problems are pathogens that have only appeared in the 30 years prior to this book being written. Globally, the most important of the newer organisms are HIV and hepatitis C, though old enemies, such as TB, Pneumococcus, and malaria, are still killing millions throughout the world. The dreadful epidemic of Ebola virus disease has shaken the world’s biosecurity.
New challenges
As we proceed through the twenty-first century, several factors are serving to ↑ the relative importance of infection over other areas of medicine. Infections such as Ebola, zika, and avian/swine influenza are continually emerging and re-emerging. Antimicrobial resistance is increasing, meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and carbapenemase-producing Enterobacteriaceae (CPE) being the most infamous examples. There are more immunosuppressed patients as a result of ↑ use of chemotherapy agents and organ transplantation. Tourists and other travellers are making their way to ever more remote parts of the world. Medical tourism is a cheap way of getting cosmetic surgery. Migration of populations has always spread disease and this continues today. There are concerns of wilful bioterrorism. All of these factors mean that the infectious differential diagnosis—even in the developed world—grows ever longer.
It is always worth bearing in mind infection in a differential diagnosis is often treatable. Accordingly, it is always better not to miss treatable options over incurable ones. Furthermore, some infectious diseases like Ebola, multidrug-resistant tuberculosis (MDR-TB), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome coronavirus (MERS-CoV), and avian/swine influenza have major public health consequences, including for the treating physician.
A challenge to the clinician
The same infection is often capable of causing a wide variety of clinical pictures. HIV, for example, is a great mimicker. Acquired immune deficiency syndrome (AIDS) is defined by its consequences. This is not so surprising, given the genetic variety of mankind, hence individual responses to a bewildering variety of infecting agents. Some clinical syndromes can be caused by many, quite different pathogens. Good examples include pneumonia, hepatitis, and endocarditis.
Furthermore, some infectious diseases can resemble non-infectious diseases. For example, amoebic colitis can resemble ulcerative colitis; syphilis can present with serious psychiatric symptomatology; a brain abscess or a tuberculoma can resemble a brain tumour; and TB of the vertebral column can resemble metastatic malignancy. Getting it wrong can be catastrophic for the patient.
Other diseases can mimic infections
Non-infectious diseases can resemble infection. Examples include gout of the first MTP joint, rather than cellulitis; cervical lymphadenopathy due to lymphoma, rather than TB; familial Mediterranean fever as a cause of PUO; SLE leading to Libman–Sachs endocarditis; adult Still’s disease as a cause of fever and neutrophilia; and inflammatory carcinoma of the ♀ breast resembling a pyogenic breast abscess.
Importance of epidemiological factors
Epidemiology is fundamental to determining which, if any, infecting agents, and therefore investigations, are relevant in a given patient.
Geography
This is very important and needs to be specific.
Some infections are common the world over and include
•Staphylococcal infection.
•Salmonella infections.
•Pneumococcal pneumonia.
•Gonorrhoea.
•Thrush (candidiasis).
•TB.
•Influenza.
•EBV.
•HIV (although sub-Saharan Africa is still the worst affected area).
•Hepatitis C.
•Herpes simplex.
•Threadworm (Enterobius).
•Syphilis.
Some infectious diseases are commoner in the tropical world, e.g.
•Malaria.
•Diphtheria (although still common in Eastern Europe).
•Rheumatic fever.
•Enteric fever (typhoid and paratyphoid).
•Hepatitis E (although increasingly widespread).
•Poliomyelitis (very restricted now).
•Rabies (although Eastern Europe has significant disease).
•Viral haemorrhagic fever (VHF), including Ebola, Lassa, Marburg.
•Onchocerciasis (river blindness).
•Schistosomiasis.
•Leishmaniasis (although commonly found around the Mediterranean).
•Ascariasis.
•Cutaneous myiasis (e.g. tumbu and bot fly).
Some infectious diseases are common in some parts of the developed world, but not in others, including
•Lyme disease.
•Babesiosis.
•Ehrlichiosis.
•Histoplasmosis.
•Hydatid disease.
•Anisakiasis.
In the USA, only certain areas are endemic for
•Lyme disease.
•Coccidioidomycosis.
•Babesiosis.
•Histoplasmosis.
•Tick-borne diseases.
Travel and vaccination history
This is important for many reasons. Travel exposes patients to new infectious agents to which they have no immunity. Immunization schedules differ throughout the world, and some groups refuse to have their children vaccinated. Vaccination before travel does not necessarily happen as recommended. The clinician must therefore be aware of the distribution of common infections. Great variation in antibiotic resistance patterns can be observed in different parts of the world; this clearly has an impact on the choice of empirical treatment. Finally, travel often has an impact on patterns of sexual and risk-taking behaviour (see Fig. 5.1).
Sexual and drug-taking activity
Searching and personal questions may need to be asked. This is sometimes difficult to do, even with great experience. Patients will/may not admit to high-risk sexual activity or the use of illegal substances and may need to be pressed. Beware of using family members as ‘translators’. The clinician must maintain high clinical suspicion at all times, even and especially when the patient does not fit a social stereotype. Bear in mind that any patient may have a ‘double life’, of which even his/her spouse is unaware. It is dangerous for the clinician to assume that being married equates to sexual fidelity or even heterosexuality.
It may not be immediately obvious that the fever, rash, and hypotension in a woman may be related to her tampon usage (toxic shock syndrome), yet menstruation can be a difficult subject to discuss in some cultural settings. TB of the ♀ genital tract may present as infertility or menorrhagia. ‘Lumpy semen’ may be indicative of Schistosoma haematobium infection, and a ‘urinary tract infection’ or a septic arthritis in a 19-year-old man may be gonorrhoea.
Social and professional
Pets, hobbies, and jobs may well be important. The patient with pneumonia and a budgerigar could have psittacosis. The tropical fish salesman with a chronic rash on his hand could have Mycobacterium marinum infection (aka ‘fish tank granuloma’). The jaundiced volunteer cleaning out canals at weekends could have leptospirosis related to contact with rats. The cat owned by the middle-aged lady with recurrent axillary lymphadenopathy may be the key to her problem of cat-scratch disease (Bartonella henselae).
Assessing the patient
The recognition of an infectious disease in a patient (or the absence of one) goes far beyond the Petri dish, the microbiology bench, and PCR testing technology. The Andromeda Strain phenomenon (with all due credit to Michael Crichton MD) should be borne in mind. The disease in front of you might be the first ever presentation or the first in a new outbreak! Almost the only significant new human diseases that will appear in the future will be infectious diseases and they will keep appearing till the end of the human species (see Fig. 5.2).
Assessment should include
•A detailed history.
•Full physical examination (including temperature).
•The generation of a differential diagnosis.
•Specimen collection before antimicrobial therapy.
•Laboratory tests.
•Non-invasive procedures (including radiological tests where appropriate).
•Invasive procedures.
•The making of a definitive diagnosis (wherever possible).
When considering the possibility of an infective process, one should always consider the basic taxonomy
•Bacteria (including primitive forms).
•Mycobacteria.
•Fungi.
•Viruses.
•Protozoa.
•Helminths.
•Prions.
•Myiasis.
Investigations available to the infectious diseases or general physician
Many tests will be performed with a view to making a diagnosis. Investigation of a patient should be rational and evidence-based, wherever possible. Although the interrogative armamentarium of the infectious diseases and tropical medicine physician is enormous—as with any other branch of medicine—the history and examination will point the way. Results will emerge which, whilst not producing a diagnosis as such, will nevertheless require following up. For example, low C5 levels in recurrent meningococcal septicaemia may need immunological assessment. (Remember also that there is a new drug eculizumab which specifically inhibits C5.) IgG deficiency leading to recurrent pneumonia may require regular infusions of γ globulins. A low CD4+ cell count, which is not due to HIV infection, could be a feature of sarcoidosis.
Making a diagnosis alone is not the only issue at stake. Some tests must be done if a patient is going to be treated safely. Examples might include: G6PD levels before administering primaquine for hypnozoite eradication in malaria; TB cultures for antibiotic sensitivity prior to starting empirical therapy; and exclusion of pregnancy before using certain antibiotics such as doxycycline and ciprofloxacin. Other tests relate to the fact that some infectious diseases are dangerous to others, including the doctor! Prime examples of this would be MDR-TB and extensively drug-resistant TB (XDR-TB), avian influenza, SARS, MERS-CoV, and Ebola, all of which are potentially dangerous for the population at large and need to be identified (or at least suspected wherever appropriate) and treated in an isolation unit.
Investigating the infectious diseases/tropical medicine case
Available diagnostic techniques
Direct detection
•Microscopy:
•Direct—e.g. faecal parasites (± iodine) or malarial and trypanosomal blood films.
•Special stains—these include Gram and ZN stains.
•Electron microscopy (EM)—for viruses and other pathogens.
•Immunofluorescence— with specific sera.
•Presence of toxin: e.g. Clostridium difficile.
•Antigen detection (
Serology, pp. 396–399).
•Molecular assays ( Molecular diagnostics, pp. 406–407): these include gene probes, amplification assays, e.g. PCR.
•Point-of-care tests: bedside rapid diagnostic tests (RDTs) for malaria, influenza, HIV, and HBV
Culture
•All body fluids and tissues can be cultured. As a general principle, the larger the sample sent, the greater the yield. It is good practice to forewarn the laboratory before sending any unusual samples, especially if there is a risk to laboratory staff, and label accordingly ( Biohazards, pp. 402–405) Laboratory preparation and specialist containers may be required. Adequate clinical details should be written on any request forms, including travel history. The choice of culture technique can vary dramatically, depending on the organism that is being sought. There may be only one chance to culture the correct organism, so it should not be wasted.
•Once a culture has grown, identification may be:
•Through special growth media, culture temperature, or atmosphere.1
•By biochemical reactions (e.g. catalase or coagulase), API strip, or matrix-assisted laser desorption/ionization (MALDI).
•With specific antisera (e.g. latex agglutination or immunofluorescence).
•Using molecular-based methods (e.g. specific probes, restriction enzyme patterns, DNA sequencing).
•Using antimicrobial susceptibility testing (e.g. metronidazole sensitivity for anaerobes and vancomycin for Gram +ves).
Serology
Serology, pp. 396–399.
Other tests as appropriate
(See appropriate sections.)
•Biochemistry.
•Haematology.
•Immunology.
•Molecular tests.
•Radiology.
•Stool and bowel contents.
•Tissue biopsy and deep aspiration specimens.
•Other tests.
Culture techniques
Microorganisms exist in nature as mixed populations. Diagnosis of an infection means identifying the relevant pathogen. Furthermore, different organisms can cause the same disease (e.g. pneumonia), require very different treatment and management, and have different prognoses. Whilst some specimens (e.g. stool, sputum) contain extremely large numbers of varied organisms, some specimens (e.g. blood, CSF, urine) should be sterile, unless infected or contaminated during their collection.
Microbiological culture assists with the aetiological diagnosis of bacterial, fungal, protozoal, or viral illness by enabling identification and susceptibility testing of the isolated organism(s). Bacterial culture was the first to evolve, but useful data on other pathogenic groups can also be obtained through the use of culture-based methodologies (although options for treatment are currently more limited for viruses and fungi than for bacteria). Furthermore, culture of mycobacteria, viruses, and fungi usually takes longer than most bacterial cultures; therefore, the data obtained are most valuable for late confirmation of the diagnosis or for epidemiological purposes (e.g. for predicting the appropriate constituents for a polyvalent influenza vaccine).
Bacteria
Three major steps are involved in extracting pure cultures from a diverse population of microorganisms and identifying a pathogen. Many of these processes can now be automated (see Fig. 5.3).
1.An isolation plate is created. To do this, the mixture must be diluted until the various individual microorganisms have been dispersed far enough apart on an agar surface, so that, after incubation, they will form visible colonies isolated from the colonies of their neighbours. Specialized culture media (such as selective media, differential media, enrichment media, and combination selective and differential media—a great many exist) may be used to supplement mechanical techniques of isolation. Culture can be aerobic or anaerobic. (Note: specimens for the isolation of anaerobic pathogens require special care, as anaerobic bacteria die in the presence of O2. Such specimens should therefore be transported in a reduced container.) Lastly temperature can be used to further select for pathogenic organisms, e.g. Campylobacter jejuni is unusual as it will grow at 41°C.
2.A pure culture is created. To achieve this, an isolated colony will be selected out and carefully ‘picked off’ the isolation plate for transferring to a new sterile medium. Following incubation, all the organisms in the new culture will be descendants of the same organism.
3.The organism can then be identified through various manoeuvres:
•Increasingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and other automated systems (see below).
•Other techniques include:
•The colony appearance and susceptibility to specific antibiotic discs.
•The microscopic appearance.
•The staining responses (e.g. Gram +ve vs Gram −ve).
•The use of a range of biochemical tests designed to uncover characteristics typical of a particular organism, e.g. catalase reaction, sugar fermentation as in the API strip. The presence of the enzyme coagulase is useful for distinguishing Staphylococcus aureus from less pathogenic coagulase −ve staphylococci (normal skin commensals).
•Less frequently now is the use of antisera (direct serology) for culture confirmation:
— Agglutination and latex agglutination tests can be used on colonies to identify Escherichia coli 0157, Streptococcus pneumoniae, serogroups of Neisseria meningitidis, Shigella, and Salmonella, Lancefield groups of β-haemolytic streptococci, and serotypes of Haemophilus influenzae.
— Detection of specific antigens by direct fluorescent antibody (DFA) staining can be used to identify colonies of Streptococcus pyogenes, Bordetella pertussis, and the species and serotypes of Legionella.
Ideally, specimens for bacterial culture should be taken before antibiotics are administered. This may not always be feasible, but the information yielded may well be less than ideal.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
MALDI-TOF MS is a new automated technique using mass spectrometry, which allows the analysis of biomolecules such as DNA, proteins, peptides, sugars, and large organic molecules, which tend to fragment when ionized. MALDI-TOF MS is a 3-step process. Firstly, the sample is mixed with a matrix material and applied to a metal plate. Secondly, a pulsed laser irradiates the sample, triggering disintegration. Finally, the analyte molecules are ionized in a plume of hot ablated gases, which are then accelerated into a mass spectrometer for analysis. The resultant spectra generated can be used for the identification of microorganisms, when compared to stored database profiles. Species diagnosis by this procedure is much faster, more accurate, and cheaper than other techniques. MALDI/TOF has become the standard method for species identification in large, modern medical microbiological laboratories. It is easy to use, robust, and rapid, allowing accurate bacterial identification of a large variety of species within a few minutes, with only a small amount of culture sample required for the analysis (104–106 CFU). The absence of the need to purify the suspect colonies allows for a much faster turnaround time. There are other new automated systems on the market such as the BD Phoenix identification method (BD Diagnostic Systems, Sparks, MD) which uses modified conventional, fluorogenic, and chromogenic reactions. Similarly, the Biologue system (Biologue, Inc.) combines up to 2000 phenotypic tests. The older Analytical Profile Index (API, bioMérieux) system uses direct inoculation into a strip of various reactants, manually producing a code which is then compared to a database. (See Fig. 5.4.)
Antibiotic sensitivity
Once a bacterium is isolated, it can be cultured in the presence of antibiotic(s) to assess if it is susceptible to that agent or not. The minimum inhibitory concentration (MIC) is the lowest antibiotic concentration at which the microorganism under assessment shows no visible growth in vitro. The MIC can provide the clinician with precise information about the infecting bacterium’s degree of antibiotic susceptibility and enable him/her to avoid antibiotics to which the organism shows resistance.
For organisms exhibiting unusual resistance patterns, susceptibility panels using methodologies such as broth microdilution, gradient diffusion, and/or disc diffusion have been created to assist clinicians.
On occasions, these data will need to be linked to testing of blood levels for some antibiotics (e.g. gentamicin, vancomycin, cycloserine). MALDI-TOF and other automated systems can also be used to identify antimicrobial resistance patterns. Nowadays genome sequencing can identify genetic mutations associated with resistance.
Viruses
Viral culture is rarely used now, but it differs significantly from bacterial culture as viruses require a very different type of medium to grow. Molecular techniques, such as PCR and antigen detection, have become more effective, can quantitate the amount of virus present, and have almost completely replaced culture.
The appropriate type of specimen to collect, the best means of transport, and the most appropriate cell culture to use will vary with the particular virus suspected, the specimen site, and the time of the year.
•The choice of specimen is very important. Numerous viruses enter via the mucosa of the upper respiratory tract, yet that virus may compromise multiple or distant tissues and organs.
•Swabs can be used to collect a variety of specimens from the body surfaces for viral detection, e.g. nose, throat, eye, skin, and rectum. A nasopharyngeal aspirate (NPA) may be the more appropriate specimen if influenza is suspected. It is important to collect mucosal cells, as this is where the virus resides. Deeper specimens, such as blood and CSF, will be appropriate for some viruses. Different viruses will need different collection approaches, e.g. heparin, citrate, and EDTA are all acceptable for the detection of CMV by PCR, antigenaemia testing, or culture, but for some other viruses, only citrate should be used if they are to be cultured.
•Unlike many bacterial or fungal pathogens, the time of year is important to keep in mind when making a diagnosis of certain viral diseases. For example, enteroviruses circulate almost exclusively in the summer months and influenza likewise circulates during the winter months.
•Timing is important when collecting specimens for viral detection. They should be collected as early as possible after the onset of symptoms, as once viral shedding ceases, culture will be impossible and serological and molecular techniques may be the only way of diagnosing the viral pathogen.
•Some viruses cannot be cultured, e.g. viral agents of diarrhoea (caliciviruses, astroviruses, and coronaviruses), HCV, and HBV.
Laboratory assays for antiviral susceptibility testing include phenotypic and genotypic assays. Genotypic assays have almost completely replaced phenotypic testing, especially for HIV and HBV. Genotyping of HCV is critical in guiding treatment. Phenotypic assays require growth of the virus in vitro and so present a biohazard. In these circumstances, genotypic assays ( Molecular diagnostics, pp. 406–407) are now routinely available and very useful.
Fungi
Unlike bacterial and viral diseases, direct microscopy can often be used to diagnose fungal infections (based on distinctive morphological characteristics of the invading fungi, e.g. Aspergillus or tinea, and/or the judicious use of special stains such as methylthioninium chloride (methylene blue)). However, histopathological diagnoses should be confirmed by culture, wherever possible. Conversely, although diagnoses are usually made by isolating the causative fungus from bodily samples, the presence of a fungus in a culture from a non-sterile site does not mean that it is pathological (e.g. Candida isolation from sputum). Fungal infection can only be definitively established with evidence of tissue invasion histologically. There are also a range of serological tests available for systemic mycoses ( Serology, pp. 396–399), but few provide definitive diagnoses by themselves. Antigen detection has been promising but generally has poor sensitivity and specificity, even when used in combination. For example, galactomannan and β-D-glucan can be indicative of invasive aspergillosis in combination with PCR for Aspergillus, but the context also needs to be considered. Allergic bronchopulmonary aspergillosis (ABPA) patients can have serum samples tested for total IgE and aspergillus-specific IgE.
An exception is cryptococcal antigen (CrAg) which is useful in serum and CSF samples for diagnosis of cryptococcal disease, especially meningitis in immunocompromised HIV patients.
Fungal culture techniques are similar to bacterial ones. They are most useful for detecting dimorphic fungi, which manifest both mycelial and yeast forms. This group includes Candida species, Cryptococcus neoformans, Blastomyces dermatidis, Histoplasma capsulatum, Penicillium marneffei, and Coccidioides immitis. MALDI can identify most fungal species.
Protozoa
Protozoa of the genera Acanthamoeba and Naegleria may cause fatal CNS disease. Acanthamoeba species are free-living amoebae associated with keratitis; they may also cause granulomatous encephalitis. Another free-living amoeba Naegleria fowleri is able to cause acute fulminant meningoencephalitis and is usually associated with a history of swimming in freshwater lakes or brackish water. In suspected cases, CSF and other suspicious clinical material may be cultured on a non-nutrient agar plate seeded with a ‘lawn’ of Gram −ve bacteria (such as E. coli). Pathogenic amoebae can be identified microscopically.
Worldwide the most important protozoan infection are the plasmodia causing malaria. They can be cultured, but this is rarely of use clinically. The mainstay of malarial identification is direct microscopy, although antigen detection tests are now available.
Immunological tests
Immunological methods are in wide usage to detect many pathogens present in clinical samples. Serology refers to the laboratory usage of antigen–antibody reactions for such diagnostic purposes. Diagnosis is made by detecting antibody or antigen in blood and/or other bodily fluids, or by the identification of pathogens in culture. More recently, interferon γ release assays (IGRA) have been developed to look for evidence of T-lymphocyte reactivity to antigens from pathogens such as Mycobacterium tuberculosis.
Both direct and indirect serological tests exist
Indirect serological techniques
Employ antigen–antibody reactions to detect specific antibodies manufactured in response to an antigen or antigens on an infecting pathogen’s surface. These antibodies are found circulating in the patient’s blood or present in other body fluids.
Direct serological techniques
Employ antibodies to detect specific antigens. Because this technique can be used to identify and type cultured organisms ( Culture techniques, pp. 390–395), not only does it have individual clinical value, but it also has important epidemiological applications.
HBV is a good example of an infection where both antigen and antibody profiles are diagnostically, therapeutically, prognostically, and epidemiologically important:
•Antibody detection of a specific antibody, e.g. anti-hepatitis Be antigen (anti-HBe antibody), anti-hepatitis B surface antigen (anti-HBs antibody).
•Antigen detection, e.g. hepatitis B ‘e’ antigen (HBeAg), hepatitis B surface antigen (HBsAg).
•More recently, pre-core mutants of HBV have emerged that are not picked up by the common antigen tests.
(See Fig. 5.5.)
Antibody tests
A wide variety of methodologies for assessing antibody response are available such as immunofluorescence, agglutination, ELISA, and complement fixation (CF).
Sub-classification of organisms, through serogrouping, can be valuable epidemiologically, e.g. whilst investigating an outbreak of meningococcal (N. meningitidis) disease; if the culprit is determined to be type C, vaccination can be utilized to control the outbreak. However, this is being replaced by molecular techniques.
General principles
(See Fig. 5.6.)
1.Specific IgM levels indicates a ‘new’ infection.
2.Specific IgG levels indicates a ‘new’ or a ‘previous’ infection, or, in some cases, immunity generated by vaccination.
3.↑ IgG (‘rising titre’) when two samples (‘paired sera’) are taken with an appropriate intervening interval between them indicates a ‘new’ infection or re-infection. Diagnosis (as indicated by seroconversion) necessitates a diagnostic antibody titre or a 4-fold ↑ in antibody titre.
4.Seroconversion is said to have occurred in situations 1 and 3.
Viral antibody tests
These can be very useful because once viral shedding has ceased, viral culture is of no further value. They include tests for HIV-1, HIV-2, human T-lymphotropic virus (HTLV)-1, HTLV-2, hepatitis A, hepatitis B, hepatitis C, δ agent (hepatitis D), hepatitis E, EBV, CMV, dengue, Ebola virus disease, Lassa fever, respiratory syncytial virus (RSV), mumps, measles, rubella, influenza, parainfluenza, St Louis encephalitis, West Nile virus, yellow fever, SARS, and many more (see Fig. 5.7).
Bacterial antibody tests
Less useful, these include anti-streptolysin (ASO) and anti-DNAse B for streptococcal infection.
They can be useful for non-culturable or difficult-to-grow organisms in the correct clinical context, e.g. cat-scratch fever (B. henselae), B. pertussis, Lyme disease (Borrelia burgdorferi), Brucella species, C. jejuni, Chlamydia species, Q fever (Coxiella burnetti), E. coli 0157, Francisella tularensis, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycoplasma pneumoniae, N. meningitidis, Neisseria gonorrhoeae, Rickettsia prowazekii, Salmonella species, Treponema pallidum (including Treponema pallidum haemagglutination assay (TPHA), rapid plasma reagin (RPR), VDRL, fluorescent treponemal antibody absorption (FTA-ABS), IgM-FTA, IgM-ELISA), Yersinia enterocolitica/Y. pseudotuberculosis (Widal test).
Protozoal antibody tests
Include tests for amoebiasis, toxoplasmosis, leishmaniasis (kala-azar), African trypanosomiasis (sleeping sickness), American trypanosomiasis (Chagas’ disease), babesiosis, and Toxoplasma gondii.
Helminthic antibody tests
Include tests for Echinococcus granulosus (hydatid disease), Echinococcus multilocularis (alveolar echinococcosis), Microsporidium species, schistosomiasis (bilharzia), strongyloidiasis, filariasis, onchocerciasis, Trichinella spiralis, Toxocara canis, Taenia solium (cysticercosis or pork tapeworm), paragonimiasis (Chinese lung fluke), and gnathostomiasis.
Fungal antibody tests
See above, but include tests for Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus versicolor, B. dermatidis, Candida albicans, C. immitis, C. neoformans, H. capsulatum, Mukorazeen.
Note: in the case of CF antibody assays for antibodies to coccidioidomycosis, these are specific and do not require proof of rising levels. They can provide indispensable confirmatory evidence for a diagnosis of coccidioidomycosis as well as an indication of the relative risk of extrapulmonary dissemination. In a case of chronic meningitis, a +ve CF for anti-coccidioidal antibodies in the CSF often provides the only definite diagnostic indication of the need for aggressive antifungal therapy.
Further reading
British Infection Association. Infection resources. 
https://www.britishinfection.org/guidelines-resources/infection-resources/.
Public Health England (2009). Public health laboratories: general clinical laboratory tests. https://www.gov.uk/guidance/public-health-laboratories-general-clinical-laboratory-tests.
Antigen tests
Antigen measurement is achieved through techniques such as CF and immunodiffusion. A variety of bodily fluids can yield diagnostically useful antigens, including saliva, serum, urine, CSF, and fresh stool. The choice depends upon the clinical context. Again many of these tests have been replaced by PCR or, in the case of HIV, combined antigen detection with antibody responses to improve sensitivity.
Viral antigen tests
Include mumps, CMV, influenza, HIV, hepatitis B and C, RSV, parainfluenza viruses, adenovirus, rotavirus, and varicella-zoster virus.
Bacterial antigen tests
Include L. pneumophila (serotype 1) and B. burgdorferi (in urine), β-haemolytic streptococci, Pneumococcus, C. difficile, H. influenzae, N. meningitidis, H. pylori, and C. jejuni.
Helminthic antigen tests
Filariasis.
Protozoal antigen tests
Include malaria, giardiasis, Trypanosoma cruzi (Chagas’ disease), Pneumocystis jiroveci (formerly Pneumocystis carinii). Malaria antigen tests form the basis for the so-called RDTs which are now the mainstay of diagnosis for malaria in both endemic and non-endemic areas. Two main forms are available; histidine-rich protein 2 (HRP-2) is specific for Plasmodium falciparum, whilst Pf LDH can distinguish between P. falciparum and P. vivax. They are rapid and almost as sensitive as PCR and critically do not need highly trained staff to carry out. They are not so reliable for the other species P. ovale, P. malariae, and P. knowelsi.
Fungal antigen tests
Include C. neoformans (CrAg), H. capsulatum, and mannoprotein antigen in C. albicans.2
Collection of specimens
Principles of good specimen collection
•Good-quality specimen and clinical information produce the most valuable data.
•Optimal time of collection, e.g. take bacterial specimens before administering antibiotics.
•Collect the optimal type of specimen wherever possible, e.g. pus is preferable to a ‘pus swab’.
•Acquire expertise in specimen collection—ensure minimal contamination by normal flora (e.g. MSU, use of a tongue depressor for throat swab collection).
•Freshness of specimens—rapid transport to the laboratory is essential (especially for anaerobic organisms and for ‘hot stools’ for parasite diagnosis).
•Collect the appropriate number of specimens at the appropriate intervals, e.g. paired antisera should be taken at least 1–6 weeks apart if a diagnostic rising titre is to be demonstrated.
•Be aware of biological hazards. It is critical to categorize samples with potential pathogens in them and label appropriately.
•Biohazard level 1: bacteria and viruses, including Bacillus subtilis, E. coli, and varicella, as well as some cell cultures and non-infectious bacteria. Precautions against the biohazardous materials are minimal, most likely involving gloves and some sort of facial protection.
•Biohazard level 2: bacteria and viruses that cause only mild disease to humans or are difficult to contract via aerosol in a laboratory setting such as hepatitis A, B, and C, some influenza A strains, Lyme disease, Salmonella, mumps, measles, scrapie, dengue fever, and HIV. Routine diagnostic work with clinical specimens can be done safely at Biosafety level 2 (BSL-2), using BSL-2 practices and procedures. Research work (including co-cultivation, virus replication studies, or manipulations involving concentrated virus) can be done in a BSL-2 (P2) facility, using BSL-3 practices and procedures.
•Biohazard level 3: bacteria and viruses that can cause severe or fatal disease in humans, but for which vaccines or other treatments exist such as anthrax, West Nile virus, Venezuelan equine encephalitis, SARS virus, MERS-Co, hantaviruses, TB, typhus, Rift Valley fever, Rocky Mountain spotted fever, yellow fever, and malaria. The parasites P. falciparum, which causes malaria, and T. cruzi, which causes trypanosomiasis, also come under this level.
•Biohazard level 4: viruses and bacteria that cause severe to fatal disease in humans and for which vaccines or other treatments are not available such as Bolivian and Argentine haemorrhagic fevers, Marburg virus, Ebola virus, Lassa fever virus, Crimean–Congo haemorrhagic fever, and other haemorrhagic diseases. Variola virus (smallpox) is an agent that is worked with at BSL-4, despite the existence of a vaccine, as it has been eradicated. When dealing with biological hazards at this level, the use of a +ve pressure personnel suit, with segregated air supply, is mandatory. The entrance and exit
of a Level 4 biolab will contain multiple showers, a vacuum room, a UV light room, an autonomous detection system, and other safety precautions designed to destroy all traces of the biohazard. Multiple airlocks are employed and are electronically secured to prevent both doors from opening at the same time. All air and water services going to and coming from a BSL-4 (P4) laboratory will undergo similar decontamination procedures to eliminate the possibility of an accidental release.
Surface specimens include
•Anal/anorectal: e.g. gonococcus (N. gonorrhoeae).
•Cervical swab: e.g. HSV, gonococcus, human papillomavirus (HPV).
•Ear swab: e.g. otitis externa, otitis media, bacterial and fungal infections.
•Foreign bodies: almost always infected if causing trouble! (Includes iatrogenic foreign bodies such as arthroplasties, cardiac valves, pacemakers, ventriculo-peritoneal shunts, etc.) Foreign bodies in the ear, nose, or vagina can lead to prolonged (and often unpleasant) discharges.
•Genital ulcers: dark ground microscopy for syphilis organisms. Also chancroid, Entamoeba histolytica.
•Indwelling catheters: include urinary catheters, IV cannulae, Portacaths, etc. If a catheter is thought to be the source of an infection, cultures should be performed, and if the catheter or cannula is removed, this can be sent for culture. Urinary catheters are always colonized by bacteria. Intravascular foreign bodies such as central venous catheters and prosthetic heart valves are often affected by bacteria which are normally non-virulent such as coagulase −ve staphylococci.
•Laryngeal swab: can be useful for TB.
•Nasal, pharyngeal, gingival, and throat: e.g. meningococcus, S. aureus carriage, streptococcal infections, pertussis, adenovirus. NPAs are useful for diagnosing upper respiratory tract infections (URTIs) such as influenza and RSV through DIF tests and, more commonly now, PCR. In lepromatous leprosy, a swab from the anterior nares may reveal acid-fast bacilli indicative of this infection.
•Ophthalmic: e.g. bacterial conjunctivitis, adenovirus, rabies (from corneal impressions.3 For trachoma, PCR, direct fluorescein-labelled monoclonal antibody (DFA), and EIA of conjunctival smears are useful.4
•Skin:
•Abscess—culture for bacteria and other unusual organisms.
•Dermal scrapings, nail clippings—fungal infections (tinea—includes pedis, capitis, cruris, versicolor forms).
•Petechial rash scrapings—meningococcus (occasionally gonococcus).
•Throat: e.g. C. albicans, diphtheria, gonococcus, croup organisms.
•Urethral: e.g. Chlamydia, gonococcus.
•Vagina (high vaginal swab): e.g. S. aureus in toxic shock syndrome (including toxin testing), Gardnerella, gonococcus.
Normally sterile fluids include
•Amniotic fluid: bacterial infection can cause premature delivery, and rDNA was detected by PCR ( Molecular diagnostics, pp. 406–407) in samples from 15 (94%) of 16 patients with +ve amniotic fluid cultures.5 Hydrops fetalis can be caused by congenital infections (e.g. CMV, parvovirus B19, toxoplasmosis, syphilis, and Chagas’ disease), and making a diagnosis may involve analysis of amniotic fluid with cultures, PCR, etc.
•Ascites: consider TB (consider laparoscopy for biopsying peritoneal lesions for culture as well as histology; Gastrointestinal tract investigations, pp. 412–413).
•Blood: multiple samplings at separate times from separate body sites may need to be taken, e.g. in endocarditis. For some organisms and pathologies, an extended period of culture may be needed.
•CSF: possibilities include, e.g. meningococcus, pneumococcus, L. monocytogenes, TB, fungi (e.g. C. neoformans), and viruses ( Tissue biopsy and deep aspiration specimens, pp. 418–421).
•Ejaculate (semen): if the semen contains a high number of leucocytes, this may be an indication of either infection or inflammation. WBCs are considered significant if >1 million found in each mL of ejaculate. Sexually transmitted diseases (STDs), e.g. gonorrhoea, or urea, plasma, and prostate infections come into the differential diagnosis. S. haematobium (bilharzia) may cause haemospermia and be found in ejaculate.6 Acute mumps orchitis can be associated with loss of spermatozoa.
•Ocular fluids (intra-): include aqueous humour and vitreous humour. Bacterial, fungal, and parasitic problems can affect the interior of the eye.
•Pericardial fluid: the commonest organisms will include staphylococci, streptococci, pneumococci, H. influenzae, meningococci, and TB.
•Pleural fluid: numerous pathologies, including underlying bacterial pneumonia, tuberculous pleurisy, parasitic infections (such as strongyloidiasis), and fungal diseases (such as histoplasmosis). Biopsy of the pleura under direct visualization by video-assisted thoracoscopy (VATS) may give a better diagnostic yield.
•Synovial fluid (joint aspirate): bacterial infections can be very destructive and the options are legion. Staphylococcal disease is the commonest, but TB must always be borne in mind. Viral arthritides are usually self-limiting, and treatment is supportive.
•Urine: standard culture and sensitivity, e.g. MSU specimen, catheter specimen of urine (CSU)—useful for diagnosing cystitis, pyelonephritis, prostatitis, etc. (prostatic massage may be helpful for improving diagnosis of prostatic infections); ‘early morning urine’ (EMU) for TB, and a terminal specimen for S. haematobium (bilharzia) best collected around midday.
Normally infected fluids include
•Pus: e.g. abscess contents, wound swab/aspirates, drainage swabs. Usually bacterial (consider both aerobic and anaerobic), but amoebic and hydatid options need to be considered when the lesion is in the liver.
•Saliva: normally contains a wide range of commensal flora. Cannulation of a parotid gland duct may yield a specific pathogen that is causing a problem in that gland.
•Sputum: includes tracheal aspirate, induced sputum (obtained with physiotherapy assistance), and bronchoalveolar lavage (BAL), which may be needed in sicker patients unable to produce sputum or in conditions where copious sputum production may not be a feature (such as P. jiroveci pneumonia (PCP) in HIV infection). C&S assists with identifying a vast range of organisms, including and especially TB (always ally sputum culture to direct microscopy).
•Stool: vast range of uses ( Gastrointestinal tract investigations, pp. 412–413). Includes direct microscopy for parasites, ova, and cysts, i.e. giardia and ascariasis, culture for Salmonella, Campylobacter, Shigella, E. coli 0157, typhoid and paratyphoid, Plesiomonas shigelloides, and enteroviruses, and antigen detection of rotavirus. ‘Hot stools’ (from patient to the microbiology bench in <1h) may be helpful for amoebae, strongyloides larvae, etc.
Further reading
Health and Safety Executive. Biological agents: managing the risks in laboratories and healthcare premises. http://www.hse.gov.uk/biosafety/biologagents.pdf.
Molecular diagnostics
These tests are now the mainstay of mainstream clinical practice. HIV viral load and antiretroviral drug resistance are considered routine tests, whilst examination of the CSF for JC virus DNA by PCR is the method of choice for the diagnosis of progressive multifocal leukoencephalopathy (PMLE). Genetic testing for the rifampicin resistance mutation in RPO provides a rapid indication of MDR-TB, e.g. Cephid GeneExpert. (See Fig. 5.8 for the immunological profile of HIV disease.)
The areas of greatest value include
•Detection and quantification of viruses to monitor and guide therapy, e.g. HCV, HIV, HBV, CMV.
•Detection of slow-growing organisms, e.g. TB, atypical mycobacteria.
•Diagnosis of pathogens, which are potentially too dangerous for the laboratory staff to handle, e.g. VHF, smallpox, avian/swine flu.
•Detection of organisms killed by antibiotics prior to culture samples being taken, e.g. meningococcal sepsis.
•Detection of organisms that cannot be cultured, e.g. HCV.
•Detection of unusual diseases, e.g. helminthic diseases, fungi.
•Detection of toxins elaborated in small quantities by bacteria, e.g. toxic shock syndrome toxins.
•Detection of mutations manifesting resistance to antimicrobial agents (genotypic resistance testing), e.g. HIV, CMV, TB.
•Elucidation of pathogens that are as yet ‘undiscovered’, e.g. 16s ribosome testing.
Available molecular techniques include
•PCR: this test uses probes to look for the presence of the genes of infecting organisms ( Polymerase chain reaction amplification of DNA, pp. 324–326).
There are numerous PCR tests available, and it is particularly valuable for hepatitis C (including for genotyping), HIV, and TB. A universal eubacterial PCR (for genus and species identification of prokaryotes) and a universal fungal PCR (genus and species identification of fungi) are available. HBV DNA quantification is accomplished through PCR.
Choosing an appropriate sample for the application of PCR testing is important (e.g. biopsy of possible Kaposi’s sarcoma lesion and Kaposi’s sarcoma-associated herpesvirus (KSHV) (human herpesvirus (HHV)-8); BAL fluid and PCP; small bowel biopsy and Whipple’s disease; CSF and meningococcal disease, HSV, or M. tuberculosis). Note: fluid samples for PCR usually have a higher yield when not spun in a centrifuge.
•LCR (ligase chain reaction): works through specific probe amplification through the use of DNA ligase. Greatest value in Chlamydia infection.
•Transcription-mediated amplification (TMA): uses an isothermal amplification system. Amplified telomerase products are rna, detected using a non-isotopic hybridization protection (HPA) system. Identification of HCV, TB, gonococcus, and Chlamydia are among its potential uses.
•Branched-chain DNA (bDNA): a signal amplification methodology able to quantify HIV RNA levels.
•Nucleic acid sequence-based amplification (NASBA): a quantitative test for HIV RNA; also of value with CMV.
•Hybridization with nucleic acid probes: detects specific ribosomal RNA and is most widely used for culture confirmation of an organism (e.g. fungi, mycobacteria).
•Sequencing: organisms are identified by direct sequencing of amplified gene fragments. Has been applied to TB, H. pylori, enteroviruses, and HIV (for assessing drug resistance). Whole genome sequencing (next-generation sequencing) is moving forward rapidly but brings with it the problems of bioinformatics and ‘big data’.
•Restriction fragment length polymorphisms (RFLPs): restriction enzymes are used to cut up DNA into pieces, and the fragments are then subjected to gel electrophoresis (such as Southern blotting; Southern blotting, pp. 322–323). The patterns produced can be used to identify organisms.
Further reading
Shanson DC.Microbiology and Clinical Practice, 3rd edn. Oxford: Butterworth-Heinemann, 1999.
Haematology
Many infectious diseases manifest haematological changes that are diagnostically valuable.
Blood film
Blood smear examination provides general data on the size and appearance of cells, as well as data on particular cell segments, whilst pathogens may be seen, e.g. malaria, African trypanosomiasis, Chagas’ disease, babesiosis, borreliosis, bartonellosis, ehrlichiosis, filaria (time of day the blood is taken may be significant in this condition), haemolysis, and evidence of hyposplenism. Thick and thin blood films should be considered, especially where malaria is concerned; at least three blood films, each taken 24h apart, should be performed. Blood films are also useful in assessing if a patient has DIC.
Bone marrow examination
Culture (for, e.g. TB, brucellosis, Mycobacterium avium intracellulare, typhoid/paratyphoid, CMV), microscopy (for, e.g. leishmaniasis), and establishing cell line integrity (e.g. WBC abnormalities). An aspirate is generally useful for culture purposes and for establishing what cells are present in the marrow, but a trephine is needed if structural information is needed (e.g. to establish if granulomata suggestive of TB are present).
Coagulation studies, fibrin degradation products, D-dimers
Useful where DIC is suspected. DIC is a common association of severe sepsis (especially meningococcal disease). Coagulation abnormalities are also present in conditions such as VHF, P. falciparum malaria, rickettsial diseases, etc. D-dimers may assist with the diagnosis of thrombosis but are generally raised in infection.
Cold agglutinins
A haemagglutination-based test. Can be caused by M. pneumoniae (most commonly), influenza A, influenza B, parainfluenza, and adenoviruses.
Differential white cell count in peripheral blood. Useful associations include:
•Eosinophilia and parasite infection.
•Neutrophilia and bacterial sepsis.
•Neutropenia and atypical pneumonias.
•Atypical lymphocytes and EBV.
•Neutropenia and pyrexia.
Erythrocyte sedimentation rate
Together with CRP ( Biochemical tests, pp. 416–417), the rate of erythrocyte sedimentation is sensitive to the extent of a body’s response to a lesion or disease. The ESR is important for pointing to the possible existence of an organic disease, but a normal result does not exclude the presence of disease. A ↑ ESR points to the need for additional investigations and, if ↓, is very useful in monitoring the course of a disease. Procalcitonin (PCT) may eventually prove to have even greater value in a similar role.7
Ferritin levels
↓ in iron deficiency, e.g. associated with hookworm infestation of the bowel (Ancylostoma duodenale, Necator americanus) or H. pylori-associated gastritis. Ferritin levels are often extremely high in Still’s disease, an important non-infective cause of pyrexia and neutrophilia. It is also markedly raised in haemophagocytic lymphohistiocytosis syndrome (HLH). Serum iron and TIBC may be helpful in a fuller evaluation ( Biochemical tests, pp. 416–417).
Glucose-6-phosphate dehydrogenase
Useful in therapy of benign malarias, e.g. P. vivax and P. ovale (a deficiency will cause severe haemolysis when primaquine is used to kill the hypnozoite phase to prevent relapse).
Hb concentration and red cell parameters (especially mean cell volume)
Useful in, e.g. anaemia of chronic infection, haemolysis, iron deficiency (microcytosis) associated with hookworm infestation of the bowel or H. pylori-associated gastritis, macrocytosis due to vitamin B12 deficiency with Diphyllobothrium latum (fish tapeworm) infestation.
Hb electrophoresis
Can detect haematological conditions such as thalassaemia, sickle-cell disease, etc. These can predispose to certain infections via hyposplenism such as salmonellosis and melioidosis.
Haemolysis screen (including reticulocyte count)
May be abnormal in, e.g. malaria, DIC, EBV, VHF, E. coli 0157 gastroenteritis due to HUS, rickettsial infections, dengue, and gas gangrene. Hp levels can be useful ( Biochemical tests, pp. 416–417), since they are generally ↓/absent in haemolysis.
Monospot test (Paul Bunnell test)
Diagnostic of EBV infection.
Sickling test
Uncovers sickle-cell disease, known to be associated with Salmonella osteomyelitis, chronic leg ulcers, etc.
Thrombocytopenia
Characteristic in some conditions, e.g. HIV disease, P. falciparum malaria, and dengue.
Vitamin B12 level
↓ in D. latum infestation, TB of the terminal ileum, etc.
Radiology
Plain X-rays
•Chest: the potential diagnoses are legion, including pneumonia, TB, pleural effusion/empyema, bronchiectasis, PCP, tropical eosinophilia and other parasite-related diseases (e.g. paragonimiasis), occupational risks for infections (e.g. silicosis and TB), and post-varicella calcification. Also useful to exclude non-infectious causes of fever such as cancer or sarcoidosis.
•Plain abdominal X-ray: e.g. bowel dilatation and perforation, calcification of adrenal glands and lymph nodes (e.g. TB, histoplasmosis), ‘babies head’ sign of schistosomal bladder calcification.
•Dental radiography (orthopantomogram): occult dental sepsis.
•Elsewhere: e.g. limbs for osteomyelitis, skeletal muscles for calcified cysticercosis lesions, joints for Charcot changes (such as in syphilis).
More sophisticated imaging
Endoscopic retrograde cholangiopancreatography
Using contrast medium and radiographs to define the anatomy of the biliary tree and pancreatic duct. Useful for HIV-associated biliary tree disease (including porta hepatis nodal lymphoma), parasites (e.g. Clonorchis sinensis, Ascaris lumbricoides), and pancreatic disease such as TB.
Intravenous urogram
Defines renal anatomy. Renal infection, such as pyelonephritis, renal calculi, malignancy, or anatomical abnormalities (including congenital) leading to recurrent infections. Computed tomography intravenous pyelography (CT-IVP) is generally considered superior now.
Magnetic resonance imaging
Including with contrast enhancement using gadolinium.
•Cranial: vCJD (exhibits bilateral pulvinar high signal), encephalitis, rabies, sagittal sinus thrombosis, PMLE. MRI is also more sensitive than CT when looking for infective SOLs such as tuberculoma, especially in and around the cerebellum.
•Bones: essential for the diagnosis of osteomyelitis.
•Spinal cord: essential for diagnosis of spinal infections and myelitis.
•Elsewhere in the body: defining solid lesions, fluid-filled lesions, etc.
•Magnetic resonance cholangiopancreatography (MRCP): non-invasively defines the hepatopancreatic biliary tree anatomy.
•New techniques: e.g. as cardiac MRI imaging, show great promise in the diagnosis of valvular disease.
Computed tomography
Including with contrast enhancement.
•Cranial: e.g. brain abscess, paranasal sinus disease, middle ear disease, orbital sepsis, cysticercosis, mastoid air cells.
•Chest: e.g. cardiac lesions (possibly with associated endocarditis risk), mediastinum (e.g. lymphadenopathy, including retrosternal), lung lesions such as bronchiectasis, lung abscess, other non-infectious pathologies.
•Abdomen: delineates intra-abdominal abscesses and abnormalities in retroperitoneal and mesenteric lymph nodes, defects in the spleen, liver, kidneys, adrenals, pancreas, and pelvis.
•CT-IVP: powerful tool for defining the anatomy of the urinary tract.
•Multislice CTPA: e.g. for defining PEs as a cause of PUO.
•CT-guided biopsy: to specifically pick out an area for sampling, e.g. liver lesion, lymph node, mediastinal mass.
•CT colonoscopy: helpful in diagnosing colonic disease, especially in older patients unable to undergo invasive testing (e.g. when looking for an associated colonic tumour in a patient with Streptococcus bovis endocarditis).
Ultrasound
•Abdomen: evidence of pancreatic, liver, renal, and biliary tree/gall bladder abnormalities (e.g. abscess, hepatic cyst, presence or absence of spleen, ascites, gallstones, etc.).
•Thoracic: pleural effusion, empyema (can assist with drainage).
•Echocardiography: to help exclude cardiac vegetations of endocarditis, TB pericarditis (with effusion), myocarditis. Note: both TTE and TOE approaches are available, each yielding data of differing value in different situations.
•Doppler studies of blood vessels: to exclude DVT such as in the legs.
•Guided biopsy: to specifically pick out an area for sampling, e.g. liver lesion, lymph node.
•Drainage: to specifically pick out an area for draining, e.g. liver abscess, pleural effusion.
Radionuclide scanning
•Has limited value but can localize area of inflammation for biopsy.
•Indium (111In)-labelled granulocyte scan: may help localize many infectious or inflammatory processes (i.e. deep sepsis).
•99mTechnetium bone scan: bone and joint sepsis.
•V/Q scan: to exclude PE as a cause of PUO, to delineate consolidation, abscess, bronchiectasis, etc.
Positron emission tomography-computed tomography (PET-CT)
Enormous potential for locating localized infective processes, especially in the brain. Increasingly used in oncology and can be helpful in PUO.
These techniques are all described in detail in Chapter 13.
Gastrointestinal tract investigations
Biopsy-based
•Duodenal biopsy (Crosby capsule and endoscopic methods ± EM): e.g. Whipple’s disease, giardiasis, cryptosporidium, strongyloidiasis.
•Gastric biopsy: H. pylori.
•Laparoscopy: useful to exclude TB and other infections in the presence of ascites (biopsies should be sent for histology and C&S; peritoneal biopsies have a higher yield than ascitic fluid).
•Liver biopsy: Tissue biopsy and deep aspiration specimens, pp. 418–421.
•Oesophageal biopsy: e.g. candidiasis, CMV (e.g. in advanced HIV disease).
•Sigmoidoscopy and bowel biopsy: e.g. amoebiasis, pseudomembranous colitis (C. difficile infections), exclusion of microscopic colitis and IBD.
Gastrointestinal contents-based
•These are rarely performed now, as endoscopic sampling is standard.
•Baermann concentration technique: the method of choice for the detection of Strongyloides stercoralis.
•Capsule endoscopy: involving the swallowing of a pill-sized capsule containing digital video recording equipment, which broadcasts to a receiver outside of the body. Enormous potential for diagnosing pathology within the GIT, including small bowel infective processes such as TB.8
•Duodenal aspirate: e.g. giardiasis, cryptosporidium, strongyloidiasis.
•Enterotest (string test): e.g. giardiasis, cryptosporidium, strongyloidiasis.
•Hot stools: little evidence that they need to be hot! ( Culture techniques, pp. 390–395.)
•Stool C&S ( Culture techniques, pp. 390–395).
•Stool microscopy: for ova, cysts, parasites (e.g. amoebae, helminths such as A. lumbricoides).
•Stool EM: good for viruses, e.g. rotavirus and norovirus, but rarely done.
•Stool chromatography: C. difficile toxin, a specific C. difficile toxin A and B EIA and PCR are used.
•Sellotape®(adhesive) strip test: for the threadworm Enterobius verminformis. To perform this test, roll some clear adhesive tape around four fingers of a hand, sticky side out, whilst an assistant spreads the buttocks. In good lighting, identify the involved perianal area, and apply the tape 1–2 times to the affected perianal area. Place the tape on a slide with the clean side downwards, trim the tape, label the slide, and send to the laboratory.
•Toxin tests: most widely used for C. difficile toxins A and/or B; the definite diagnosis of botulism food poisoning is the examination of faeces for Clostridium botulinum and toxin (EMG is also helpful). In wound botulism (e.g. in drug injectors), the organism may grow in material taken directly from the wound(s); also used for E. coli 0157.
Gastrointestinal tract function
•D-xylose absorption test: for malabsorption syndromes such as Whipple’s disease and tropical sprue.
•13C breath test: for detection of H. pylori.9
•Faecal elastase: for diagnosis of pancreatic exocrine insufficiency in conditions such as HIV
•Vitamin levels: diagnosis of fat-soluble vitamin malabsorption in pancreatic or small bowel disease or of B12 levels in terminal ileal problems such as with TB.
Immunology
Cutaneous hypersensitivity tests are discussed elsewhere ( Other tests, pp. 422–423).
•Complement (especially ‘terminal’ complements C5–C9): deficiencies can lead to recurrent meningococcal sepsis, pneumococcal disease, etc.
•Cytokine studies: currently experimental, but levels of interferon can sometimes be helpful.
•Differential white cell count ( Haematology, pp. 408–409): neutropenia and lymphopenia are associated with bacterial sepsis.
•Igs: deficiencies lead to recurrent infections (some cases may be hereditary, and family history is important). Levels may also be higher—IgM tends to be markedly ↑ in brucellosis, malaria, trypanosomiasis, and toxoplasmosis.
•Splenic dysfunction: indicated by a history of surgical removal (not always clear!) or a condition associated with hyposplenism (e.g. coeliac disease/dermatitis herpetiformis, sickle-cell disease), an abnormal blood film, and an absent spleen on abdominal imaging. This state may be associated with recurrent meningococcal infection and life-threatening pneumococcal sepsis. Once diagnosed, the patient will need appropriate vaccinations and advised to always carry a warning card and/or wear a MedicAlert® bracelet or similar.
•T-cell subsets: the absolute CD4+ (T4) cell count and the CD4+/CD8+ (T4/T8) cell ratio is of value. HIV disease, TB, and sarcoidosis are associated with reduced CD4+ cell levels, HIV with a reversed CD4+/CD8+ cell ratio.
•Human pharmacogenomics:
•HLA typing—HLA B5701 is associated with hypersensitivity (sometimes fatal) to the antiretroviral drug abacavir, and so patients should be tested before prescribing this drug.
•IL-28R polymorphisms—these can be associated with treatment failure in hepatitis C infection.
Biochemical tests
A number of biochemical tests are useful in the diagnosis and assessment of a range of infectious illnesses.
•AFP: ↑ in hepatocellular carcinoma (associated with HCV and HBV). Note: much higher AFP than in other causes of hepatocellular damage.
•ABGs: assessment of sepsis, assessment of pneumonia.
•Ca-125: ↑ false +ve in peritoneal TB.
•CRP: together with the ESR, a valuable method for monitoring infections. CRP is an acute phase reactant, ↑ in bacterial infections and ↓ in viral infections. PCT may be of value.
•Creatinine phosphokinase (CPK) level: ↑ in L. pneumophila infection.10 Also ↑ with zidovudine (AZT) usage in HIV disease.
•Glucose metabolism: DM is a common association of infection, particularly TB. Consider performing a fasting glucose level, an OGTT, or checking HbA1c levels.
•Hp levels: part of the haemolysis screen ( Haematology, pp. 408–409). Iron levels (serum iron), TIBC: iron ↓ (TIBC ↑) in iron deficiency, e.g. associated with hookworm infestation of the bowel (A. duodenale, N. americanus) or H. pylori-associated gastritis. Serum ferritin may be helpful.
•Lactate levels: may be ↑ in HIV-associated mitochondrial toxicity syndrome. Also high in severe sepsis syndrome.
•Lipid abnormalities (cholesterol, TGs): HIV drug toxicity.
•LFTs: abnormalities are present in many conditions, e.g. hepatitis, leptospirosis, yellow fever, antimicrobial drug toxicity (e.g. in TB).
•ALP: in the serum of healthy adults, ALP mostly originates from the liver. Biliary obstruction, often associated with sepsis, leads to an ↑ in serum concentration of ALP.
•Bilirubin: determination of bilirubin levels (conjugated and unconjugated) is important in the differential diagnosis of jaundice.
•γGT: ↑ in serum concentration of γGT is the most sensitive indicator of liver damage.
•Pancreatic amylase level: ↑ with pancreatitis in, e.g. mumps (consider also salivary amylase), toxicity with antiretroviral drugs (e.g. didanosine or DDI).
•Pleural fluid analysis: analysis for LDH levels is useful (as well as albumin, total protein, and amylase). An exudate, which implies infection in the differential diagnosis, is defined by at least one of the following criteria: pleural fluid/serum total protein ratio >0.5, pleural fluid/serum LDH ratio >0.6, or pleural fluid LDH > two-thirds the upper limits of normal of serum LDH.
•Pregnancy test: some infections, e.g. varicella, genital herpes (simplex), and TB, are often more serious in pregnancy. The use of some antibiotics, e.g. ciprofloxacin and tetracyclines, is relatively contra-indicated in pregnancy. There is the potential to prevent vertical transmission of HIV if diagnosed prior to delivery.
•PCT: shows promise for detecting and following ‘inflammation induced by microbial infections’.10
•Serum Na+levels: hyponatraemia associated with legionnaires’ disease11 but may also be related to intracranial sepsis or hypocortisolaemia.
•Synacthen®test: TB and histoplasmosis can damage the adrenal glands, leading to an Addisonian state (hypocortisolaemia).
•Vitamin D levels: if deficient, may predispose to TB and may lead to difficulties with treating TB infections due to hypocalcaemia (consider checking levels in patients with darkly pigmented skins, especially those with a culture of wearing clothing over most of their body surface). Deficiency may be exacerbated by some anti-infective drugs, notably rifampicin and antiretrovirals.
Tissue biopsy and deep aspiration specimens
Whatever part of the anatomy from which they are taken, biopsy specimens should be evaluated both histopathologically (with specialized stains used, wherever appropriate) and by culture for bacteria, mycobacteria, fungi, viruses, and prions (using specialized culture techniques, where appropriate).
Bone marrow biopsy
( Haematology, pp. 408–409)
Important for TB, brucellosis, Mycobacterium avium intracellulare typhoid/paratyphoid, leishmaniasis.
Cerebrospinal fluid
Whilst the main objective of an LP is usually to obtain fluid for microscopy and C&S, there are other useful tests that can be performed. The opening pressure should be between 10 and 20cmH2O—infective and other processes may alter this. Whilst the usual approach to obtaining CSF is through an LP, if the pressure is high, a cisternal puncture can be performed instead (normally the advice and help of a neurosurgeon would need to be sought). In neonates, foramenal puncture is a possibility. A CT scan is usually performed beforehand to assess the risk of ‘coning’, although it is by no means a guarantee.
Along with the Gram staining process and microscopy, other tests to consider include a complete blood cell count and differential measurement of glucose and protein levels, ZN staining for TB, and bacterial, mycobacterial, viral, and fungal cultures. On occasions, other tests that might be considered include:
•Wet mount (for amoebae such as Acanthamoeba; Culture techniques, pp. 390–395).
•PCR for HSV, herpes varicella-zoster, enteroviruses, TB, JC virus, and cryptococcosis ( Molecular diagnostics, pp. 406–407).
•Antibodies to specific pathogens (e.g. arboviruses; Serology, pp. 396–399).
•India ink capsule stain (for cryptococcosis).
•CrAg ( Serology, pp. 396–399).
•VDRL, etc. for syphilis ( Serology, pp. 396–399).
•14-3-3 protein—specific protein marker present in the CSF of patients with vCJD.
•Xanthochromia to help exclude SAH also seen in leptospirosis.
•Cytology to help exclude carcinomatous meningitis.
•Assessing comparative CSF protein–cellular levels if GBS (recognized association of infections such as Campylobacter gastroenteritis) is being considered.
Liver biopsy
Indications are numerous and include assessment of viral hepatitis (especially HBV and HCV, including possible cirrhosis and/or hepatocellular carcinoma), assessment of PUO (including TB and lymphoma), and
determining if the patient has a medication-induced liver disease. Only on <1% of occasions does the liver biopsy overestimate the amount of hepatic damage. Fibroscan is increasingly used in conjunction with blood tests to non-invasively score liver damage, but it is not as accurate as the biopsy.
The biopsy is commonly preceded by an USS examination of the liver to determine the best and safest biopsy site. Usually, the biopsy is conducted under ultrasonic guidance to avoid major blood vessels. Coagulation status should be optimized at the time of biopsying, including by the administration of clotting factors.
The risks of the traditional liver biopsy (not performed under ultrasound guidance) include
•Pain (1 in 5 patients).
•Haemorrhage (1 in 500 patients).
•Bleeding requiring transfusion or surgery (1 in 1000 patients).
•Pneumothorax and/or puncture of the gall bladder, kidney, or bowel (1 in 1000 patients).
•Death (1 in 5000 patients).
Equivalent figures are not available for USS-guided liver biopsy, but the technique is well established. Liver biopsy material should be subjected to microbiological culture, as well as to histological assessment.
Lymph node sampling
The likely pathologies depend upon whether or not lymphadenopathy is regional or generalized, and upon the site.
•Biopsy: for histology and culture, especially for TB, for tropical infections such as chancroid, and for other relevant infections such as cat-scratch fever (B. henselae). If regional, the differential diagnosis varies with the site; if intra-abdominal, for example, TB, Y. enterocolitica, and adenovirus should be considered.
•FNA: useful as full biopsy for culture purposes, but no structural information available (similar to the aspirate vs trephine issue in BM sampling), therefore difficult to exclude lymphoma from differential.
Respiratory samples
Sputum tests
•Microscopy: can perform direct microscopy (e.g. for Aspergillus species, eggs of paragonimiasis), Gram stain, ZN, PCP (silver staining needed).
•Induced sputum: e.g. for TB, PCP, Aspergillus.
•Tracheal aspirate: used in ill individuals. May produce similar material.
Bronchoscopy
•BAL: useful for TB and other mycobacteria, PCP, fungi, melioidosis, resistant bacteria (e.g. Pseudomonas), RSV, and paragonimiasis.
•Lung biopsy: useful for TB and other mycobacteria, PCP (needs silver staining, immunofluorescence test (IFT), or PCR), fungi, melioidosis, resistant bacteria (e.g. Pseudomonas), RSV, and paragonimiasis. Also for exclusion of non-infective causes of non-resolving pneumonia.
Open lung biopsy
When not feasible to obtain intrathoracic tissue by less invasive means.
Pleural disease: effusion, empyema, biopsy
Consider, e.g. TB, pneumococcal sepsis, underlying neoplasm (and rarer conditions like strongyloidiasis). Biochemical analysis of pleural fluid can help ( Biochemical tests, pp. 416–417). An empyema will have white cell count, protein, pH, LDH changes compatible with an exudate, and possibly organisms visible and/or culturable within the fluid. A pleural biopsy can be obtained blindly with an Abraham’s needle, but in recent times pleuroscopy and VATS has developed into a better option.12
Skin biopsy
Biopsy and hair sampling
•Useful for, e.g. TB, Kaposi’s sarcoma (caused by HHV-8 and associated with HIV), onchocerciasis, the aetiology of warts (common viral warts vs molluscum contagiosum—the distinction can be important in view of the therapeutic options and the potential for malignant change in some sites such as the ♀ cervix).
•The identification of pathogenic arthropod parasites, e.g. myiasis (the invasion and feeding on living tissues of humans or animals by dipterous larvae such as that of the tumbu fly), scabies, lice, ticks, and chigger fleas, depends on the offending agent being seen and correctly recognized or the appropriate specimen (e.g. excision biopsy) being taken and examined histologically.
Skin snips
•Filarial infestations: examination of skin snips will identify microfilariae of Onchocerca volvulus and Mansonella streptocerca. Skin snips can be obtained using a corneal–scleral punch, or more simply a scalpel and needle. The sample must be allowed to incubate for 30min to 2h in saline or culture medium, and then examined microscopically for microfilariae that would have migrated from the tissue to the liquid phase of the specimen. In onchocerciasis, nodulectomy is also of value, as is examination of the eye with a slit lamp.
•Leprosy: acid-fast bacilli are present in the skin.
Other tissues and collections are numerous and include
•Bone infection/abscess/osteomyelitis: consider, e.g. pyogenic sepsis, TB, atypical mycobacteria, sickle-cell disease, ectopic ova of schistosomiasis. History is important, e.g. with a history of fight trauma to a hand, anaerobic bony infection may be more likely.
•Brain lesions and abscesses: biopsy and drainage useful for, e.g. TB, herpes simplex, rabies, cysticercosis, encephalitis, vCJD, JC virus, and toxoplasmosis (in HIV infection).
•Cervix: HPV, HSV, N. gonorrhoeae.
•Joint infections: aspirate synovial fluid and consider, e.g. pyogenic sepsis, TB. An acute attack of gout (diagnosed through identifying the birefringent crystals of sodium urate) can mimic an acute infective
arthritis (including a systemic inflammatory response with neutrophilia) and should be excluded.13
•Liver abscess: consider Streptococcus milleri, hydatid disease, amoebic dysentery, necrotic hepatocellular carcinoma in hepatitis C or hepatitis B, obstruction of the biliary tree by A. lumbricoides or liver flukes such as C. sinensis.
•Muscle biopsy:
•Cardiac—may point towards myocarditis or Chagas’ disease.
•Skeletal—may be used to identify parasites, including, e.g. trichinosis, cysticercosis.
•Nerve biopsy: peripheral nerve biopsy (e.g. posterior auricular nerve) may reveal tuberculoid leprosy.
•Ocular:
•Vitreous humour—e.g. intraocular infections, including fungal, HSV, herpes varicella-zoster, pyogenic bacterial.
•Cornea—e.g. rabies, CJD.
•Retina—e.g. herpes varicella-zoster, toxocariasis, CMV.
•Paranasal sinus aspirates: e.g. bacteria, fungal (such as mucomycosis).
•Pericardial biopsy: particularly important for establishing a diagnosis in chronic pericarditis, e.g. TB, fungal.
•Peritoneal infection: via laparoscopic tissue sampling and ascites sampling ( Gastrointestinal tract investigations, pp. 412–413).
•Splenic aspiration: useful in the diagnosis of visceral leishmaniasis (kala-azar) by microscopic examination and culture and demonstration of the organism.14
•Tonsillar biopsy: of particular value for diagnosing vCJD: also consider MRI scanning ( Radiology, pp. 410–411), EEG, and 14-3-3 protein in the CSF.15
Other tests
Antibiotic plasma concentration monitoring
Some drugs are toxic if the plasma levels rise too high and their use is futile if the levels are too low. Monitoring serum drug levels ensures that plasma drug levels remain within the therapeutic range. Antimicrobial drugs that may require this approach include gentamicin, vancomycin, teicoplanin, kanamycin, amikacin, tobramycin, chloramphenicol, streptomycin, cycloserine, amphotericin, flucytosine, voriconazole, and itraconazole.
Cardiac
•ECG: serial ECGs can be of value in Lyme disease, rheumatic fever, pericarditis, myocarditis, and toxic shock syndrome. Also of value in conditions where the cardiac conduction mechanism has been damaged, e.g. Chagas’ disease (American trypanosomiasis) and with a valve root abscess in severe infective endocarditis. In cholera and enteric fever (typhoid and paratyphoid), the cardiac rate will often be slower than one might anticipate for the degree of fever.
•Echocardiography: TTE and TOE for the diagnosis of endocarditis. TOE especially for prosthetic valves.
Dermatological tests
•TB skin tests: measure delayed hypersensitivity. The Mantoux test usually involves the intradermal injection of 10 tuberculin units of purified protein derivative (PPD), and the response is quantified. The reaction is read at 48–72h. Most useful epidemiologically, their individual clinical value being relatively limited. Multiple puncture techniques (the Heaf and Tine tests) are likely to be more convenient for large group studies. Interpretation of these tests is more difficult in patients inoculated with the bacillus Calmette Guérin (BCG) vaccine, and IGRAs (e.g. QuantiFERON®) are generally more helpful as they distinguish between T-cell responses to BCG and TB.
•Histoplasmin test: a +ve intradermal skin reaction to histoplasmin (the histoplasmin test) may be the only sign of past infection with H. capsulatum. Main value is epidemiological. A similar skin test exists for C. immitis.
•Mazzotti (DEC) test: for filariasis. Rarely used now, this test relied on the intense pruritic response induced by microfilariae after treatment with the antifilarial agent diethylcarbamazine* (DEC). Used in a minute quantity, it can nevertheless be associated with side effects, ranging from mild discomfort, fever, headaches, and intolerable pruritus to tachypnoea, tachycardia, and even pulmonary oedema.
Note: (*) diethylcarbamazine is not licensed for use as a filaricide.
•Skin testing for antibiotic allergy: this can be performed in the same way as for other allergens by patch testing or skin prick testing.
Narcotics and anabolic steroids screen
If +ve, these may point towards occult drug use and a concomitant risk of blood-borne viruses (e.g. HIV, hepatitis C, hepatitis B). vCJD has been transmitted through anabolic steroid injecting. The antiretroviral efavirenz interferes with these tests producing a false +ve.
Neurological
•EEG: may help with making a diagnosis of encephalitis (e.g. in patients with HSV encephalitis, the EEG may exhibit focal unilateral or bilateral periodic discharges localized in the temporal lobes), of brain abscess, or of cerebral cysticercosis. It may also be of value in vCJD.
•LP: material for C&S can be obtained, but much additional information is also gathered, e.g. the opening pressure is usually elevated in infections ( Tissue biopsy and deep aspiration specimens, pp. 418–421).
•EMG: offers rapid bedside confirmation of the clinical diagnosis of botulism. It shows a pattern of brief, small, abundant motor unit potentials. In GBS (associated with Campylobacter gastroenteritis), EMG may be helpful in excluding 1° muscle disease.
•NCS: helpful with diagnosing neuropathies (e.g. HIV, leprosy, GBS).
Ophthalmology
•Slit lamp examination: can help with the diagnosis of infective and parasitic ocular problems, e.g. uveitis (syphilis, Reiter’s syndrome), O. volvulus larvae, toxocariasis, toxoplasmosis, candidiasis.
•Colour vision testing (Ishihara): used to assess toxicity associated with ethambutol in treatment of TB.
•Direct and indirect ophthalmoscopy: essential for the diagnosis of CMV retinitis in all HIV +ve patients with a CD4 cell count <100.
Pulmonary
•Pulmonary function tests: bronchial hyper-reactivity can be assessed for (often provoked by infection, e.g. ABPA) and interstitial lung disease checked for (which can include, e.g. TB, fungal infections, etc.).
Clinical investigation in action: pyrexia of uncertain origin
Common problem in hospital medicine, with huge potential differential diagnosis.
PUO is best defined as a body temperature of 38.3°C centrally (rectally) for 3 weeks or longer without the cause being discovered, despite extensive investigation for at least 1 week.
Assessment should include
Observation of fever pattern
Some conditions, such as VHF, typhoid, and malaria, may exhibit characteristic fever patterns.
Complete and repeated detailed history, with emphasis on the recognized differential diagnosis, including
•Travel history.
•Antimalarial usage.
•Vaccination history.
•Past use of medical services in foreign parts may be especially important (e.g. blood transfusions, splenectomy post-trauma, needlestick assaults).
•Drug-using history (including illicit drugs and especially injecting).
•Exposure to certain agents and/or animals (e.g. pet ownership, occupational risk of animal contact such as veterinary medicine, nursing, farming, meat packing).
•Hobbies (e.g. caving is linked to histoplasmosis and canal fishing to leptospirosis).
•Sexual history (and risk taking).
•Menstrual history.
Complete and repeated physical examination, including re-evaluation of previous findings, e.g.
•Check the skin, eyes, nail beds, lymph nodes, heart, and abdomen.
•A new sign, e.g. cardiac murmur, may have developed over time.
•The judicious use of repeated tests is also critical, depending upon the context.
•Laboratory and radiological tests, taking into account new data, e.g. blood cultures, blood films, autoantibody screen, radiological findings.
•Non-invasive procedures, taking into account new data, e.g. genito-urinary assessment such as high vaginal swab.
•Invasive procedures, e.g. liver biopsy, BM biopsy, laparoscopy, Waldeyer’s ring assessment by an otolaryngologist.
Common groups of causes of a PUO in an adult are
•Infections.
•Connective tissue diseases.
•Occult neoplasms (especially leukaemia, lymphoma, and renal carcinoma).
A list of relevant pathologies might include
HIV, TB, endocarditis, osteomyelitis, malaria, syphilis, zoonoses (e.g. brucellosis, Lyme disease, tularaemia), viral hepatitis (especially hepatitis C and B), typhoid/paratyphoid, pelvic inflammatory disease, chronic meningococcaemia, dental sepsis, tumours such as lymphoma, renal carcinoma, liver metastases, familial Mediterranean fever, multiple PEs, drugs, rheumatological, e.g. Still’s disease, TA, SLE, granulomatosis with polyangiitis (GPA, previously known as Wegener’s granulomatosis), vasculitis, atrial myxoma, factitious fever, Munchausen’s syndrome, Munchausen’s syndrome by proxy.
With improved non-invasive and microbiological techniques, most cases of PUO are found not to be caused by infections, but rather by other systemic diseases such as sarcoidosis, SLE, and TA. However, there are also infectious diseases capable of causing prolonged fever that should always be considered and factored into the assessment because they are often treatable and/or transmissible to others and will have serious consequences if missed. A definitive diagnosis is not made in around 25% of patients; however, they tend not to come to any harm when observed over a long period.
Endocarditis
Endocarditis is a deep-seated infection that behaves like a deep-seated abscess. Indeed, an abscess can form adjacent to an infected cardiac valve or shunt. The diagnosis involves thoughtful clinical assessment, including whether or not there is a history of injecting drug use, and requires multiple blood cultures and cardiac assessment. The Duke criteria form the basis of the diagnosis.16 Assess clinically for likelihood, e.g. background of injecting drug use, congenital heart disease, prosthetic valves, rheumatic fever, scarlet fever. Endocarditis may manifest changing cardiac murmurs over a period of time, as well as a number of additional signs.
•Establish diagnosis: echocardiography (especially TOE)—to look at valves, cardiac chambers, shunts, etc.
•Establish aetiology:
•Blood cultures (multiple)—consider culturing for unusual organisms such as fungi, HACEK organisms (Haemophilus species, e.g. H. parainfluenzae, H. aphrophilus, and H. paraphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella species), L. monocytogenes, etc.
•Serology—Q fever (Coxiella burnetii) phase I and II, C. albicans.
•Assess clinical status:
•ECG—tachycardia, conduction abnormalities.
•CXR—cardiac size, PEs with right-sided endocarditis.
•U&E—to assess renal compromise, if any.
•Haematology—WBC.
•Inflammatory markers—ESR, CRP.
•Proteinuria—to assess renal compromise, if any.
•Blood-borne virus status—HIV, hepatitis C, hepatitis B if there is a history of drug injecting.
•Assistance with therapy:
•Antibiotic sensitivity testing.
•Serum antibiotic levels (e.g. gentamicin, vancomycin).
•Prevention: dental assessment—for prevention in the future. Endocarditis warning card. MedicAlert® bracelet.
Tuberculosis
Consider pulmonary vs extrapulmonary disease and other epidemiological parameters. (Pleural disease is by definition extrapulmonary.)
•Establish diagnosis/aetiology:
•Radiological evidence.
•Bodily fluids (e.g. sputum, EMUs, gastric washings, CSF) and biopsies—always consider performing induced sputum, even with a normal CXR; histology may show caseating granulomata (see Fig. 5.9).
•PCR testing.
•Mantoux test.
•IGRAs such as QuantiFERON®.
•Ca-125 levels—abdominal TB in women.
•Assess clinical status:
•Inflammatory markers—ESR, CRP.
•T-cell subsets—low CD4+ cell count characteristic.
•Body weight.
•HIV testing—now essential in all patients.
•Glucose metabolism—may be an association (fasting glucose, OGTT, HbA1c).
•Assistance with therapy:
•Antibiotic sensitivity testing.
•Serum antibiotic levels (e.g. cycloserine).
•LFTs.
•Skin testing.
•Calcium and vitamin D levels.
•Gene probes (for rifampicin resistance).
•Prevention: notify cases to public health authorities. Contact tracing.
•TB and biopsies: when a biopsy is being obtained of any organ or tissue, the possibility of extrapulmonary TB should be borne in mind. If histology is performed, caseating granulomata may be seen, and appropriate staining for acid-fast bacilli (such as the ZN stain) may reveal the presence of TB organisms. However, because of the ↑ risk of MDR-TB and XDR-TB, material should always be sent to the microbiology laboratory and appropriate cultures for TB should be set up, both for diagnostic and for drug sensitivity purposes. Molecular techniques, including gene probes and PCR, are now essential parts of the diagnostic armoury for TB. In many instances, having a biopsy taken is an unpleasant experience for a patient, and remembering to perform a TB culture at the outset may prevent the patient from having to undergo an unpleasant procedure more than once.
Malaria (fever in the returning traveller)
Always consider malaria in the febrile individual returning from overseas. A detailed geographical history and malaria prophylaxis history is essential. Always consider the possibility of a coexistent second diagnosis (especially in P. falciparum infestation) such as Salmonella septicaemia (the so-called ‘algid malaria’).
•Establish diagnosis/aetiology:
•Thick and thin blood films—perform three (each 24h apart).
•Antigen tests—such as ParaSightF® and OptiMAL®.
•Blood sugar—hypoglycaemia is common and needs immediate treatment.
•Platelet count—thrombocytopenia suggestive of P. falciparum.
•Haematology—WBC.
•Inflammatory markers—ESR, CRP.
•Assess clinical status:
•Blood cultures—to exclude 2° infection.
•Haemoglobinopathy—to assess for sickle-cell disease.
•Assess the very ill patient thoroughly for markers of severity (includes LFTs, blood film for haemolysis, coagulation status, CXR, ECG, ABGs, glucose levels, lactate levels, conscious level, etc.). Note that severe P. falciparum malaria can present as a diarrhoeal illness.
•Assistance with therapy:
•G6PD levels.
•Tests of hearing—deafness can occur with quinine.
•Prevention: avoid blood donation for 18 months.
Jaundice (acute)
Jaundice can be pre- or post-hepatic, or a combination of both. Epidemiological factors are important (drug injecting, travel, unsafe food, unsafe sex, job, hobbies, vaccination history, alcohol, prescribed medications, herbal remedies, etc.). The patient may have an acute exacerbation of a chronic disease, e.g. hepatitis C. Always remember Courvoisier’s law (a distended gall bladder in a patient with obstructive jaundice means cancer) and Charcot’s triad (the characteristic presentation of acute cholangitis, with biliary colic, jaundice, and spiking fevers with rigors). Haemolysis may lead to jaundice without liver disease being present.
•Establish diagnosis:
•LFTs—conjugated and unconjugated bilirubin levels.
•Urinalysis.
•Stool examination—colour, flushability.
•Haemolysis screen—blood film, coagulation studies, antiglobulin test, etc.
•Hepatobiliary US—serial scans can assess hepatobiliary status sequentially.
•AFP levels—may suggest hepatocellular carcinoma associated with hepatitis C and hepatitis B infection.
•Establish aetiology:
•Serology—e.g. hepatitis A through to E, EBV, CMV, toxoplasmosis, leptospirosis, hantavirus, yellow fever.
•Blood culture.
•Stools for ova, cysts, and parasites—e.g. C. sinensis, ascariasis).
•Monospot for EBV.
•Hepatobiliary US—obstruction by malignancy or parasites, liver parenchyma status, gallstones.
•ERCP/MRCP—may diagnose parasitic invasion of the biliary tree, CMV/cryptosporidial disease, porta hepatis lymphadenopathy associated with HIV, etc.
•Paracetamol levels.
•Assistance with therapy:
•HIV testing—if appropriate; co-infection with HIV, HCV, and HBV an ↑ problem worldwide.
•Ethanol assessment—γGT levels, ↑ MCV.
•Molecular tests—PCR testing for HCV, circulating DNA levels in HBV.
•Antigens—hepatitis B.
•Prevention:
•Notify cases to public health authorities; safe sex education; safe drug-injecting education possible once viral diagnosis of HCV, HBV, and/or HIV established.
•Assess family, sexual partners, etc. for possible infection (HIV, HBV, HCV) and/or need to vaccinate (HBV).
•Vaccination strategies: HBV, HAV as appropriate.
Diarrhoea
Diarrhoea can be acute vs chronic, or acute on chronic. For example, a gastroenteritis illness may uncover pre-existing IBD (such as Crohn’s disease) or malabsorption (such as coeliac disease or pancreatic insufficiency). Drugs such as opiates can lead to ‘overflow’ diarrhoea. Also bear in mind that where there is one bowel pathogen, another one might be present. Antibiotic resistance is common among some bowel pathogens. Diarrhoea can appear infective but, for example, might be endocrine in origin (e.g. carcinoid syndrome, Zollinger–Ellison syndrome, medullary carcinoma of the thyroid), whilst the possibility of bowel cancer must always be borne in mind. Note that the presence of S. bovis in blood cultures may be indicative of the presence of bowel cancer until proven otherwise. Irritable bowel disease is being increasingly diagnosed. Malaria can present as diarrhoea.
•Establish diagnosis:
•Examine stools.
•Keep a stool chart on the ward.
•Establish aetiology:
•Stool C&S.
•Stool microscopy for ova, cysts, and parasites.
•Faecal fat and elastase for pancreatic insufficiency.
Pneumonic illness
Pneumonia is multi-aetiological. If recurrent, this throws up certain diagnostic possibilities that must be considered. Many epidemiological considerations are important such as travel history, occupation, pet keeping, hobbies, sexual activity, etc. Osler’s triad of rigors, pleuritis, and rust-coloured sputum is said to be characteristic of pneumococcal pneumonia.
•Establish diagnosis/aetiology:
•CXR (or CT chest).
•Serology—atypical pneumonia organisms (L. pneumophila, M. pneumoniae, C. burnetii, Chlamydia psittaci), hantavirus, RSV, influenza.
•Sputum—including induced sputum, bronchoscopy, and BAL: microscopy and culture.
•Blood cultures.
•Serum Na+level—↓ in Legionella.
•Antigen—pneumococcal (blood), Legionella (urine).
•NPA for viral culture—RSV, influenza.
•Cryoglobulins—e.g. M. pneumoniae.
•Molecular—various PCR tests for viruses and Mycoplasma.
•HIV test—essential in risk groups.
•Assess clinical status.
•(CURB65 score):17
•ABGs.
•Confusion, Urea, Respiratory rate, Blood pressure.
•US of the chest—if effusion developing (drain if necessary), check pH of fluid.
•Pulmonary function tests if appropriate.
•Assistance with therapy:
•Antibiotic sensitivity testing.
•If recurrent: consider TB testing (see earlier), HIV testing, Ig levels (to check for deficiency), assessing for hyposplenism, checking terminal complement levels (C5–C9).
•Prevention:
•Notify appropriate cases to public health authorities (e.g. Legionella, TB); isolate as necessary.
•Vaccination strategies: influenza, Pneumococcus, Hib.
•Stop smoking, if relevant.
Meningitic illness (headache and photophobia)
Meningitis can be extremely serious, particularly bacterial, mycobacterial, fungal, and protozoal forms, but viral meningitis is generally less serious. Meningitic infection is often mimicked by much less serious infections such as UTI (especially in women), throat infections (ASO, Monospot), atypical pneumonias, and sinusitis (especially ethmoidal, sphenoidal). A similar picture can also be generated by SAH. Meningococcal infection can be life-threatening without ever causing meningitis. If bacterial meningitis is recurrent, certain diagnostic possibilities must be considered. Brain abscess (consider injecting drug use, congenital heart disease, immunodeficiency, etc.) and, under certain circumstances, encephalitis can present in a similar fashion to meningitic illnesses. Where the patient has a marked petechial rash and a history of travel to Africa, Ukraine, or South America, even VHF (particularly the Congo–Crimean variety) comes into the picture (see Fig. 5.10).
•Establish diagnosis/aetiology:
•LP/cisternal puncture/foramenal puncture (in neonates)—for CSF pressure, microscopy, bacterial and mycobacterial culture (including special cultures, e.g. for Listeria), viral culture, biochemistry (e.g. protein, glucose), differential cell count, viral PCR, xanthochromia, India ink stain, CrAg testing.
•CT scan of head—sometimes necessary to exclude an SOL prior to performing an LP; CT is little better than a clinical assessment in the exclusion of raised ICP. When ↑ ICP is found, cisternal puncture and foramenal puncture is possible in skilled hands.
•CXR and assessment for atypical pneumonia, if appropriate.
•NPA (see earlier).
•Petechial rash sampling—aspirate material from a fresh purpuric lesion using a small-needle insulin syringe, Gram stain, and culture.
•Molecular—meningococcal PCR (blood and CSF), pneumococcal PCR (blood and CSF).
•Serology—urine and blood for CrAg; blood for pneumococcal antigen; urine and saliva for mumps antigen; ASO, antibodies to mumps, EBV, Cryptococcus, N. meningitidis.
•Nasopharyngeal swab for meningococcus.
•Stool for enteroviral culture.
•Monospot test for EBV.
•Assess clinical status:
•CT/MRI scan of head—assess for raised ICP; exclude SAH (xanthochromia), sagittal vein thrombosis; exclude skull fracture, especially of cribriform plate or middle ear (this can lead to recurrent pneumococcal meningitis—if there is a nasal drip, test fluid for glucose to exclude presence of CSF as CSF contains glucose).
•Differential WBC in blood.
•Inflammatory markers—ESR, CRP.
•Coagulation screen and platelet count: for meningococcal sepsis.
•ABGs—to assess acid–base balance in severe cases.
•Synacthen®test ( Short Synacthen® test, p. 225)—adrenal failure in severe meningococcal sepsis (Waterhouse–Friderichsen syndrome).
•HIV test—suggested by some pathologies and may be the overall underlying problem.
•TB assessment—may be the underlying pathology.
•Assistance with therapy:
•Antibiotic sensitivity testing.
•Serum antimicrobial levels, e.g. amphotericin, flucytosine.
•Prevention:
•Notify relevant cases to public health authorities; isolate as necessary.
•Vaccination strategies—meningococcus, pneumococcus, influenza, Hib.
•History of skull fracture—may need neurosurgery, etc.
Urethritis (with or without haematuria)
Pain on micturition can simply represent a UTI or there may be an STD, such as gonorrhoea, present. The patient’s sexual and travel history is important. Renal calculi can produce clinical pictures resembling an infection, as can dermatological conditions such as Stevens–Johnson syndrome. UTIs are commoner during pregnancy. Prostatitis can be a problem in older men.
•Establish diagnosis/aetiology:
•Urine collection (MSU)—culture (bacterial infections), microscopy (parasites, etc., such as schistosomiasis—use terminal specimen), molecular techniques (LCR for Chlamydia).
•STDs and pelvic inflammatory disease—perform high vaginal swab and urethral swabs; screen for gonococcus (includes throat and anal swabs).
•Calcular disease—exclude with urine microscopy, radiology, etc.
•Prostatitis—prostatic massage, CrAg.
•TB—can present like any other UTI.
•Reiter’s syndrome—slit lamp examination of the eye, urine, and stool culture/LCR for Chlamydia.
•Assess clinical status:
•Biochemistry—exclude renal failure (urea, creatinine, etc.).
•Markers of inflammation—CRP, ESR.
•White cell count.
•Check all other mucosal surfaces of the body (mouth, conjunctivae, nose, etc.) to help exclude Stevens–Johnson syndrome.
•Assistance with therapy:
•Pregnancy test.
•PSA to exclude prostatic carcinoma (recurrent UTIs in older men).
•Radiology of renal tract—US, IV pyelography (IVP) (to exclude underlying renal tract anatomical problems, TB involvement, calculi, etc.).
•Prevention:
•History of unsafe sex, recent new sexual partner, drug injecting—consider VDRL, HIV, viral hepatitis testing.
•TB—notify, contact trace, etc.
•Calculi—exclude hypercalcaemia, hyperuricaemia, etc.
Red, painful swollen lower leg
One of the most difficult things in medicine is to distinguish effectively between a distal DVT and cellulitis—and a combination of both! Less commonly, a ruptured Baker’s cyst of the knee can present in almost the same way. The key is in the history. Sometimes the problem is in the tissues, and sometimes in the joints (even gout and pseudogout can look like cellulitis) or the bone (osteomyelitis). Ulceration may be present on the legs. Venous and arterial insufficiency may complicate the picture—infected legs in older people can be very difficult to treat with antibiotics alone. Recent long-haul air travel may point more towards thrombosis, but swollen legs with compromised veins can easily get infected. Although rare, syphilis, yaws, and Mycobacterium ulcerans can cause leg ulcers that are potentially amenable to treatment. Pyoderma gangrenosum can resemble infection of the leg but is associated with non-infectious systemic diseases. Check for eschar from tick bite.
•Establish diagnosis/aetiology:
•Exclude DVT with Doppler US (and possible embolic disease on occasions).
•Swabs: from ulcers, between the toes usually not helpful.
•Blood cultures.
•Rarely VDRL.
•Joint assessment: urate levels for gout; assess (if relevant) for pseudogout, rheumatological screen, synovial fluid analysis (if relevant), Lyme disease titres (depends on the travel history, etc.).
•Leg ulcers in the young: consider sickle-cell disease, hereditary spherocytosis.
•Assess clinical status:
•White cell count.
•Inflammatory markers—ESR, CRP.
•Assess blood vessel integrity—e.g. compression US for venous problems, lower limb arteriography.
•Assistance with therapy:
•Exclude diabetes, HBA1c.
•X-ray, bone scanning, MRI—is osteomyelitis present?
•Prevention:
•Treat diabetes, if present.
•Treat other underlying conditions, if present.
•Patient advised to take care in future (e.g. DVT avoidance whilst travelling).
Vesicular rash
Many vesicular rashes are infective; many are not. In particular, the distribution of the rash should be carefully assessed, and joint assessment and management with a dermatologist are often valuable. If atopic, eczema herpeticum comes into the picture. Staphylococcal impetigo can cause vesiculation. If there is a relevant travel history, rickettsial pox and monkey pox come into the picture. Erythema multiforme, which often has an infective basis but can also be produced by medications, can produce a vesiculating rash (so check the mouth, eyes, and genitalia, and determine the
medication history). Non-infective blistering conditions include dermatitis herpetiformis (coeliac disease), pompholyx, and pemphigus.
•Establish diagnosis/aetiology:
•Vesicular fluid—PCR, EM, IFT, culture, etc.
•Serology—HSV, herpes varicella-zoster, rickettsial pox, Coxsackie virus, ASO titre.
•Assess clinical status:
•CXR—chickenpox (if compromised, ABGs will be needed).
•EEG—if cerebral symptoms present (e.g. cerebellar encephalitis can occur with herpes varicella-zoster).
•Monkey pox, smallpox, rickettsial pox—the patient will be ill and will require full assessment, even possibly intensive care.
•Assistance with therapy:
•Pregnancy test: herpes varicella-zoster a bigger problem in pregnancy.
•Prevention:
•Avoid precipitants with erythema multiforme.
•Manage atopy optimally.
Further reading
Chin J (ed.). Control of Communicable Diseases Manual, 18th edn. Washington DC: American Public Health Association, 2004.
Cohen J, Powderly WG (eds.). Infectious Diseases, 2nd edn. St Louis: Mosby, 2003.
Cook GC, Zumla AI (eds.). Manson’s Tropical Diseases, 22nd edn. Philadelphia: WB Saunders, 2008.
Mandell GL, Douglas JE, Dolin R, Bennett RE (eds.). Principles and Practice of Infectious Diseases, 7th edn. London: Churchill Livingstone, 2009.
Peters W, Pasvol G.Color Atlas of Tropical Medicine and Parasitology, 5th edn. St Louis: Mosby, 2001.
Shanson DC.Microbiology and Clinical Practice, 3rd edn. Oxford: Butterworth-Heinemann, 1999.
Steffen R, Dupont HL, Wilder-Smith A.Manual of Travel Medicine and Health, 3rd edn. Ontario: BC Decker, 2007.
1 Todar’s Online Textbook of Bacteriology. http://textbookofbacteriology.net.
2 Falci DR, Stadnik CMB, Pasqualotto AC. A review of diagnostic methods for invasive fungal diseases: challenges and perspectives. Infect Dis Ther 2017; 6: 213–23.
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