66  Diagnostic procedures

R. C. B. Slack

KEY POINTS
• A central aim in choosing antimicrobial therapy is to cost. This guidance is reflected in the local
match the most appropriate agent to the specific antibiotic policy.
microbial aetiology. In practice, this is constrained by • The laboratory will assist in modifying or initiating
urgency of the situation, the diagnostic information therapy by determining in vitro susceptibilities and by
available and policy decisions about antimicrobial use monitoring antimicrobial drug concentrations in
that are made on a local basis consistent with national specific circumstances. These analyses may lead to
policies. modification of therapy.
• The initial decision on whether or not to give an • Certain organisms such as streptococci and anaerobes
antimicrobial and on the agent chosen is generally have predictable susceptibilities, whereas others,
made before microbiological information is available such as enterobacteria, must be monitored
but, where possible, after microbiological samples have continuously.
been taken. • Prophylactic antimicrobials are recommended in highly
• In the UK, local and specific guidance is generally defined settings and following precise regimens. Ad
available for chemotherapy of common infective hoc usage is to be avoided.
syndromes. The strategy reflects the likely causal • Antimicrobial combinations may be given to increase
agents and their local susceptibility patterns, the efficacy, extend the spectrum of cover or prevent the
need to control antimicrobial resistance, and development of resistance during therapy.

The role of the laboratory in assisting clinicians in the of the laboratory. For further details of laboratory
diagnosis of infection is illustrated in the specimen methods, including specimen containers and culture
flow diagram shown in Figure 66.1. The choice of media used, the reader is encouraged to consult the
specimen depends on following the principles outlined recommended reading section.
in Chapter 65. The microbiology laboratory requires
enough information on the request card accompany-
ing the specimen to use the optimal methods necessary COLLECTION OF SPECIMENS
for identification of potential pathogens in particular
infective syndromes. At the most basic level, it is Samples for microbiological examination need to be
obvious that, when a swab is received in the labora- carefully collected, if possible without contamination
tory, it is necessary to know if it comes from the with commensals or from external sources. Some
throat or the vagina! But additional information is points to remember with specimens from individual
also essential: is the patient being investigated for sources are shown in Table 66.1. It is essential to use
phar­yngitis or diphtheria; for vaginal discharge or sterile containers which are leak-proof and able to
septic abortion? Furthermore, the specimen must be withstand transportation through the post if neces-
obtained with care and transported to the laboratory sary. It is more convenient for both the clinician and
without delay in an appropriate manner. The value of microbiologist if the laboratory provides request
the result is in direct proportion to the attention given cards, containers and an efficient transport system.
to these details, as well as to the skill and efficiency There is a need for staff to be aware of safety

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66 DIAGNOSIS, TREATMENT AND CONTROL OF INFECTION

Request Provide all relevant details appropriate reading. However, an outbreak of gas-
trointestinal disease inevitably leads to the question of
identifying the source and comparing strains isolated.
When disease is due to preformed toxins, as with sta-
Collection of Appropriate and well collected;
specimen correctly labelled phylococcal food poisoning, faecal examination is
unhelpful, and the diagnosis can only be made by
testing the food. Frequently, the offending item
Transport Rapid and safe
has been discarded and the examination of food
and water related to specific patients is often unre-
warding. Routine sampling of water sources and
Match card to sample;
potentially contaminated food such as poultry at
Reception record and assign
unique number various critical points of production is essential in
maintaining good public health. As part of the inves-
tigation of an outbreak it may be necessary to collect
Macroscopic
and microscopic
Initial report samples under controlled conditions which can be
(telephone) used as evidence in any prosecution. Laboratories
examination
need to be able to receive and process such specimens
in an approved way.
Antigen detection;
Preliminary report
nucleic acid
(same day) TRANSPORT
probes
Many microbes may perish on transit from the host’s
body to a laboratory incubator. Some contaminants,
Cultivation, Preliminary identity especially coliforms, may overgrow the pathogen and
isolation (day after receipt)*
so mask its presence. These two constraints make it
essential that any material for cultivation of microbes
is transported as quickly as possible to the laboratory
Identification; in a manner expected to protect the viability of any
Final report
antimicrobial pathogens. Such problems may be minimized by the
(2nd day)
sensitivity testing
use of antigen or gene probe detection because of the
relative stability of the chemical structures identified
on dry swabs.
Further identification: The ideal situation is to bring the patient to the
typing; toxin production; Supplementary report
further sensitivities
laboratory for specimen collection or take the labora-
tory to the clinic. Both approaches are used for special
*Viruses, fungi and some bacteria may take longer purposes but are obviously inconvenient for many
patients and inappropriate and costly for complicated
Fig. 66.1  Steps in the isolation and identification of pathogens from techniques such as virus isolation which need special-
an infected patient.
ized (and safe) facilities.
To overcome any drawbacks due to delay in
regulations and for all parties to understand who has reaching the microbiology department the following
responsibility for each step of the process and how methods may be used:
to minimize handling by untrained people. Special
precautions required for ‘high-risk’ specimens need to 1. Transport media. See Table 66.2.
be defined by the laboratory and hospital manage- 2. Boric acid. The addition of boric acid to urine at
ment. Storage of clinical material must be separate a concentration of 1.8% (v/w) will stop bacterial
from food and drugs, and this may necessitate provi- multiplication but lower concentrations are
sion of additional refrigerator space and transport ineffective and higher ones may kill the pathogen.
facilities. 3. Dip slides. These provide a convenient way of
inoculating urines at the clinic. They comprise
small plastic spoons or strips holding a thin layer
Food, water and environment
of agar which is dipped into the urine and then
The examination of nonclinical specimens is beyond put in a screw-topped bottle for transport. The
the scope of this book and readers are referred to agar adsorbs a fixed volume of urine and, after

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 Diagnostic procedures 66

incubation, colony counts of bacteria give a may not survive whereas certain organisms,
semiquantitative estimation of numbers. notably listeriae, flourish at low temperatures.
4. Refrigeration. Storage at 4°C before processing 5. Freezing. Temperatures of −70°C or below, which
will prevent multiplication of most bacteria. can be achieved in liquid nitrogen or special deep
However, delicate microbes such as neisseriae freezes, will preserve many microbes, providing

Table 66.1  Some important points to remember in the collection of specimens for microbiological examination

Type of specimen Comments

Respiratory secretions
Nasal swab (anterior) Only for carriage of staphylococci and streptococci
Nasopharyngeal swab For pertussis and meningococci
External ear swab Wide range of microbes, including fungi
Myringotomy and sinus samples As for abscesses – including anaerobes
Throat (pharyngeal) swab Specify if only for streptococci; mention if diphtheria possible; use special
transport media for virology
Saliva Used for antibody detection (gingival swab); otherwise discard
Laryngeal swab Specify if for mycobacteria
Expectorated sputum Often poorly collected; specify mycobacteria, legionellae, pneumocystis
Transtracheal aspirate, bronchoscopy specimens, lung biopsy Specify likely diagnosis; ask for specific tests
Pleural fluid Treat as pus; always look for mycobacteria

Gastrointestinal specimens
Vomitus Only for virology
Gastric washings For mycobacteria (particularly in children)
Gastric biopsy For Helicobacter pylori
Duodenal/jejunal aspirates Protozoa (Giardia lamblia, microsporidia, etc.)
Liver aspirates As for pus (anaerobes); consider amoebae
Spleen puncture For Leishmania spp.
Rectal biopsy Schistosomiasis
Rectal swab Only for gonococci and chlamydia
Colonic biopsy Histopathological diagnosis of amoebiasis, pseudomembranous colitis
(Clostridium difficile)
Colonic scrapings Protozoa; amoebic trophozoites (deliver to laboratory immediately)
Faeces Specify possible diagnosis; ask for clostridial toxins, parasite examination if
suspected
Peri-anal swab For eggs of threadworm

Urine
Mid-stream (MSU) Suitable for most patients
Clean catch Infants and elderly – increased contamination
Suprapubic aspirate Infants and neonates
Ureteric/bladder washout To localize infection
Prostatic massage Collect samples before, during and at end of micturition
Terminal urine Schistosome ova, chlamydia DNA amplification
Complete early morning or 24-h urine Mycobacteria (tubercle)

Central nervous system

Cerebrospinal fluid by spinal tap For meningitis collect sample for protein and glucose – test blood sugar
simultaneously, specify virology, fungi or syphilis serology
Ventricular tap Specify if through an indwelling shunt or catheter
Brain abscess As for pus (include anaerobes)

Skin and soft tissue

Skin scraping/nail clipping Dermatophyte fungi
Skin swab Rarely valuable without pus
Skin snips Onchocerciasis – seek advice
Vesicle fluid Suitable for electron microscopy for viruses
Wound swab Obtain pus if possible; record site
Pus, tissues, aspirates Describe site and any relevant operative details

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66 DIAGNOSIS, TREATMENT AND CONTROL OF INFECTION

Table 66.1  Continued

Type of specimen Comments

Genital
Urethral swab Pus for gonococci, scrape for chlamydia
Vaginal swab (adult) Candida, trichomonas, bacterial vaginosis or chlamydia DNA
Vaginal swab (prepubertal) State age; caution required if abuse possible
Cervical swab Separate media for chlamydia
Ulcer scrape Immediate dark-ground microscopy; separate media for virology or chancroid
Uterine secretions Specify puerperium or post-abortion
Pelvic aspirates As for pus
Laparoscopy specimens Include chlamydia specimen

Eye
Conjunctival swab Separate virology; scrape for chlamydia
Aspirates As for pus

Blood
Culture Strict aseptic technique; take large sample in special media before antibiotics
Bone marrow Valuable for leishmania, mycobacteria, brucella
Film Malaria (thick and thin), filaria, borrelia, trypanosomes
Whole blood Filaria (day or night samples as appropriate)
Serum antigen Rapid diagnosis of many microbial diseases (e.g. hepatitis B)
Serum antibody Retrospective diagnosis of common viral diseases, syphilis and other selected
infections; need rising titre or specific IgM

Table 66.2  Types of transport medium

Type of organism Medium Comments

Bacteria Stuart’s semi-solid agar Contains charcoal to inactivate toxic material

Anaerobic bacteria Various systems, including gassed-out tubes Not widely used, but essential for some strict anaerobes
and anaerobic bags
Viruses Buffered salts solution containing serum Contains antibiotics to control bacteria and fungi
Chlamydiae Similar to viral transport medium, but without Chlamydial antigen media contain detergent to lyse infected cells
agents that inhibit chlamydiae
Protozoa, helminths Merthiolate–iodine–formalin Kills active protozoa, but preserves cysts and ova in a form suitable for
concentration and microscopy

they are protected by a stabilizing fluid such as When parts of the specimen are separated from the
serum or glycerol. original bottle (e.g. after centrifugation of serum), the
laboratory number becomes the only recognizable
identification. Transcription errors are far more
RECEPTION common than is supposed and are especially impor-
tant for requests for human immunodeficiency virus
The importance of good documentation cannot be (HIV) or syphilis serology, which may have disastrous
overstressed. No matter how well the specimen was consequences and medicolegal implications if wrong
taken, transported and processed in the laboratory, results are given.
the end result depends on communication between
people. The clinician making the request must give
complete details on the request card and specimen EXAMINATION
to reduce errors. Staff receiving specimens in the labo-
ratory must match them with the cards and record Looking at clinical material with the naked eye or
them into a book or computer. This is usually done hand-lens should be part of the examination of a
by assigning a unique number to each specimen patient at the bedside. Sadly, many doctors delegate
and labelling both the specimen and the request. this to the laboratory staff. Many unnecessary

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laboratory tests could be avoided if unsuitable insufficiently sensitive to make a microbiological diag-
specimens were rejected on the ward or in the general nosis. Some microbes are so fastidious or slow-
practitioner’s surgery. These would include: crystal- growing that useful information cannot be given to
clear urine from patients with ‘cystitis’; well-formed clinicians. The isolation of many viruses requires
stools from patients with ‘diarrhoea’; and mouth laborious tissue culture methods that are too slow to
washings or saliva from patients with respiratory influence patient management. Microscopy is usually
symptoms. of low sensitivity, e.g. the threshold of detection of
It was once common practice to carry out certain acid-fast bacilli in sputum is about 105 microbes per
basic investigations in ward side-rooms, and kits millilitre. Microscopy also has low specificity; for
offering ‘near-patient testing’ are becoming available. example, Gram staining of faeces would yield millions
However, there are cogent arguments against bedside of Gram-negative rods, but it is not possible to recog-
pathology, including issues of safety, time, compe- nize those that are pathogenic by this means.
tence and quality control. To overcome these deficiencies probes have been
developed which combine a part that reacts with a
specific microbial structure and a part that will
Microscopy
produce a signal (colour, fluorescence or radioactiv-
Microscopy is an important part of the examination ity) after the reaction. Many microbes are detectable
of many specimens. For bacteriology, the Gram in this way. Antibodies labelled with fluorescent mol-
and acid-fast (Ziehl–Neelsen or Auramine) stains are ecules are widely used in diagnostic virology, e.g. for
usually sufficient, but for the demonstration of fungi respiratory secretions to find respiratory syncytial
or parasites special stains or concentration techniques virus. Enzyme-linked antibodies are available as
may be required. ‘Wet’ mounts, i.e. unstained prepa- enzyme-linked immunosorbent assay (ELISA) kits
rations of fluid material, are widely used in looking for chlamydia detection, and many other immuno-
at cells in urine, cerebrospinal fluid (CSF), faeces probes are commercially produced or under investi-
and vaginal secretions. None of these procedures gation. The explosion in molecular biology has
takes more than 5 or 10 min and all are inexpensive led to the widespread availability of DNA and RNA
in reagent costs and capital equipment. They are probes, which have changed from complicated
therefore ideal rapid methods and new diagnostic research techniques requiring radioisotopes to
techniques have to be judged against microscopy. An relatively simple methods that can be carried out
initial report, such as ‘Gram-negative diplococci and with minimal expertise. Use of amplified nucleic
pus cells seen’ from meningitic CSF, can be issued acid tests (e.g. polymerase chain reaction, PCR) has
within minutes of receiving the specimen and will aid greatly increased the sensitivity of probes. Provided
the clinician in confirming the diagnosis and starting a unique sequence of nucleotides is used, the method
appropriate antibiotics. is highly specific. None the less, as the new technol-
Similarly, a rapid diagnosis of falciparum malaria ogy becomes more commonplace traditional methods
can be lifesaving. Indeed, suspected pyogenic menin- will not be discarded. It is more likely that it will
gitis and falciparum malaria are among the few complement them.
con­ditions for which it is clearly justifiable to call One of the chief advantages of probes – their spe-
upon emergency laboratory services outside normal cificity – is also one of their major disadvantages. For
working hours. example, in the investigation of diarrhoea, it would be
Electron microscopy (EM) requires more elaborate impossibly laborious to have to use a separate probe
preparation than does light microscopy and it is for each of the possible microbial causes. Further-
therefore much slower. It was valuable in the diagno- more, use of probes leaves little scope for detection of
sis of certain viral infections, including viral diarrhoea the unexpected and hampers the discovery of previ-
but the availability of antigen (ELISA) and molecular ously unsuspected aetiological agents of disease. The
probes has largely replaced EM. The nonspecific advent of ‘chip’ technology (microarrays) may over-
nature of electron microscopy gives it an advantage in come this deficiency (see Ch. 6).
that any type of viral agent, if present in sufficient
numbers, may be recognized.
Serology
In situations in which microbial isolation is impossible
Nonculture methods
and probes are unavailable, evidence of infection
There are many situations where isolation of microbes may be obtained by finding a rise in antibody titre or
in vitro or in tissue culture is impossible or is the presence of specific IgM. Serology is still used

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66 DIAGNOSIS, TREATMENT AND CONTROL OF INFECTION

for diagnosing the causes of ‘atypical’ pneumonia

Table 66.3  Examples of bacterial identification
(mycoplasmal pneumonia, psittacosis, Legionnaires’ in medical laboratories
disease), syphilis, brucellosis and many viral infec-
tions, including HIV. It is preferable to take a blood Extent of identification
sample early in the illness (the acute serum) and
Reason for request Example 1 Example 2
another 10–14 days after the onset (the convalescent
serum); a four-fold or greater rise in antibody titre in Test of sterility Bacteria present
the second specimen is diagnostic of acute infection. Initial blood culture Gram-negative rod Gram-positive cocci
With some infections, such as HIV and hepatitis B report
and C, much longer times must elapse. Urine examination ‘Coliforms’ Staphylococci
Wound swab Escherichia coli Staphylococcus aureus
Outbreak E. coli O157 Staph. aureus phage
epidemiology type 80/81
Isolation of micro-organisms Pathogenicity tests E. coli O157 vero Staph. aureus
The basis of the study of medical microbiology was toxin-producing enterotoxin A-positive
laid over a century ago by the isolation of microbes
in pure culture outside the host animal. The methods
used by the fathers of bacteriology have been adapted,
simplified and, in some cases, automated for the unnecessary. Shortcuts must be made to satisfy the
modern diagnostic laboratory. The principles remain clinical demand of a final report which the doctor can
the same: use sterile equipment and media (with cell understand and which is available in time to influence
lines if necessary) and add clinical material. After management of the patient. In practice, most labora-
incubation at 37°C, for a variable time, from a few tories use simple and incomplete methods of identifi-
hours for enterobacteria to weeks for mycobacteria cation, depending on the level of useful information
and some viruses and fungi, a visible effect will be required. Typing of isolates is for epidemiological or
produced. This might be colonies growing on agar or other special reasons, and this is usually done in
a cytopathic effect (CPE) in tissue culture. The skill national reference centres using standardized methods.
of microbiology is in identifying the microbes respon- Examples of the extent of identification are shown
sible for the effect. in Table 66.3. This shows that the same organism
There are limits to the methods that can be used in may not be identified even to the genus level, or it may
a routine hospital laboratory to isolate microbes. The have extensive genetic investigation depending on
choice of media used is dictated by the specimen and the reason for the request. In a cost-conscious climate
by the clinical condition of the patient. For example, you get what you pay for and what the service thinks
in some areas of the world there is little point in you need!
looking for Corynebacterium diphtheriae in every
throat swab, so that the specific media needed are not
routinely used. It is therefore incumbent on the doctor
Antimicrobial sensitivity testing
to make sure that the laboratory is alerted to look for One advantage of good bacterial identification is that
diphtheria bacilli in any suspicious case. Similarly, it helps greatly in choosing antibiotics and in some
clinicians need to know which microbes are routinely situations in-vitro tests of susceptibility may be unnec-
sought from particular specimens so that they can essary. However, the widespread occurrence of bacte-
make a special request if they suspect the unusual. rial resistance, even in genera such as Neisseria and
‘New’ causes of illness are often found and new Haemophilus in which sensitivity to β-lactam antibiot-
methods of investigating old diseases regularly appear ics was previously assumed, has meant that tests are
on the market. It is a difficult decision for the labora- usually performed on all significant clinical isolates of
tory manager to assess at what point a ‘new’ pathogen bacteria. The relevance of information generated in
becomes sufficiently important to be routinely sought, the extremely artificial conditions of the laboratory is
or to balance the advantages of new (and expensive) discussed in Chapter 67.
technology against cheap and well tried techniques As there are technical problems in carrying out sen-
especially in resource poor countries. sitivity tests at the same time as the primary culture,
the report will be delayed for at least 24 h after isola-
tion of the pathogen. Tests of slow-growing bacteria
Identification
such as mycobacteria take much longer and tests
The full identification of each microbe isolated involving fungi, viruses and protozoa are not ordin­
in a clinical laboratory is both uneconomic and arily available. In general, all patients with acute

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 Diagnostic procedures 66

symptoms will receive treatment before the report

Box 66.1  List of notifiable diseases in England and Wales
returns to the doctor so that the result will merely
confirm that correct treatment had been chosen. • Acute encephalitis/meningitis
Sometimes, the laboratory report will allow specula- • Acute infectious hepatitis
tive antimicrobial therapy to be modified, e.g. by • Acute poliomyelitis
• Anthrax
allowing one or more drugs in a precautionary mixture • Botulism
to be discontinued. • Brucellosis
A frequent difficulty facing microbiologists is • Cholera
which, out of a rapidly increasing number of agents, • Diphtheria
• Dysentery (amoebic or bacillary)
should be tested and, of those tested, which should be
• Enteric fever (paratyphoid/typhoid)
reported. Most laboratories test a few representative • Food poisoning
compounds from among the many penicillins, cepha- • Haemolytic uraemic syndrome
losporins, aminoglycosides, tetracyclines, quinolones, • Infectious bloody diarrhoea
etc., and restrict those reported to the clinician to two • Invasive Group A streptococcal disease and scarlet fever
• Legionnaires’ disease
or three agents selected for their appropriateness in • Leprosy
the particular infection. In this way, institutional anti- • Malaria
biotic policies are reinforced and the impact of the • Measles
promotional activities of pharmaceutical companies is • Meningococcal septicaemia
• Mumps
lessened.
• Plague
• Rabies
• Rubella
REPORTS OF RESULTS • SARS
• Smallpox
• Tetanus
Just as it is important to obtain as accurate a result • Tuberculosis
as possible, so it is vital to transfer the information • Typhus
rapidly to the user of the laboratory in a form that is • Viral haemorrhagic fever
easily intelligible. Laboratory workers need to tailor • Whooping cough
• Yellow fever
their reports to suit their customers and to recognize
their needs. Trained microbiologists are used to the Health Protection Regulations 2010.
vagaries of microbial taxonomy and the profusion of
antibiotics with similar names, but physicians and sur-
geons are easily confused by laboratory jargon and
changes in nomenclature. On the other hand, clini- NOTIFICATION OF INFECTIOUS DISEASES
cians rightly expect laboratory reports to be explicit
and helpful. As in so many spheres of health care, a In addition to making a clinical diagnosis and treating
spirit of mutual respect and cooperation is in the best the individual there is a need with some infectious
interests of the patients, and this is most likely to diseases to determine the source and prevent further
happen if microbiologists regularly visit the wards and spread. In some countries there are public health laws
if clinicians are encouraged to discuss problems with specifying the method of reporting these. The list of
laboratory staff. notifiable diseases in England and Wales is shown in
In these days of computers and facsimile machines Box 66.1. This reporting system requires a clinical
it is attractive to use the latest technology to transfer diagnosis, with or without laboratory confirmation,
results, but the personal touch is essential, particularly which is notified separately. In England and Wales
for important results such as positive blood or CSF each health district has a consultant responsible for
cultures. Clinicians dealing with difficult problems communicable disease control, who is usually the
enjoy the reassurance that the laboratory has found ‘proper officer’ for the local government authority.
the cause of a patient’s illness and advice on manage- Notifications are sent to the Health Protection Agency
ment is generally well received. Seeing the problem (HPA), previously the Communicable Disease Sur-
for oneself and discussing it with the responsible veillance Centre (CDSC).
doctors is the ideal. The telephone is a satisfactory There is also an obligation, which in some circum-
substitute in many cases and is far preferable to a stances may be statutory, for laboratories to report
report form printed by a computer and arriving after isolates of certain pathogens to central authorities for
the crisis is over. It behoves us all to communicate surveillance purposes. For HIV/AIDS infection the
better and faster. system is voluntary and confidential, yet over 90% of

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66 DIAGNOSIS, TREATMENT AND CONTROL OF INFECTION

cases are reported in the UK. In an outbreak, or to prevent further spread of infection. In particular, if
where the pathogen is highly infectious, it is essential there is any indication that the infection may have
for the head of the laboratory to inform both the clini- been acquired by staff working in the laboratory this
cian looking after the patient and local public health must be reported to the relevant authority; in the UK
staff. In this way a coordinated approach can be made that is the Health and Safety Executive.

RECOMMENDED READING

American Society for Microbiology: Manual of Clinical Microbiology, Johnson FB: Transport of viral specimens, Clinical Microbiology Reviews
ed 10, Washington, DC, 2011, American Society for Microbiology. 3:120–131, 1990.
Forbes BA, Sahm DF, Weissfeld AS: Bailey and Scott’s Diagnostic
Microbiology, ed 10, St Louis, 2002, Mosby.

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