Download Basic Lab Procedures in Clinical Bacteriology 2nd ed (WHO, 2003) WW PDF

TitleBasic Lab Procedures in Clinical Bacteriology 2nd ed (WHO, 2003) WW
TagsMedical
LanguageEnglish
File Size2.3 MB
Total Pages175
Table of Contents
                            Cover
Contents
Introduction 1
Quality assurance in bacteriology 2
PART I Bacteriological investigations 19
	Blood 20
	Cerebrospinal fluid 25
	Urine 30
	Stool 37
	Upper respiratory tract infections 60
	Lower respiratory tract infections 66
	Sexually transmitted diseases 76
	Purulent exudates, wounds and abscesses 	86
	Anaerobic bacteriology 98
	Antimicrobial susceptibility testing 103
	Serological tests 122
PART II Essential media and reagents 141
	Introduction 142
	Pathogens, media and diagnostic reagents 143
Selected further reading 154
Index
                        
Document Text Contents
Page 2

A

Basic
laboratory
procedures
in clinical
bacteriology

Page 87

79

BACTERIOLOGICAL INVESTIGATIONS

A

Gonococcal colonies may still not be seen after 24 hours. They appear after 48
hours as grey to white, opaque, raised, and glistening colonies of different
sizes and morphology.

Identification of Neisseria gonorrhoeae

A presumptive identification of N. gonorrhoeae isolated from urogenital spec-
imens is based on a positive oxidase reaction and a Gram-stained smear
showing Gram-negative diplococci. Confirmation of the identification can be
obtained by carbohydrate degradation assays or other tests, using methods
and media discussed extensively elsewhere.1

Antimicrobial susceptibility testing

There is considerable geographical variation in the susceptibility of N. gonor-
rhoeae strains to benzylpenicillin. In some areas, such as sub-Saharan Africa
or South-east Asia, most gonococcal strains are now b-lactamase-producing.
Chromosomally mediated resistance to benzylpenicillin not based on b-
lactamase production is also becoming more common in many countries.
However, the disc-diffusion test is not reliable in detecting such strains.

In areas where benzylpenicillin, ampicillin or amoxicillin is still used for the
treatment of gonococcal infections, N. gonorrhoeae isolates (particularly from
cases of treatment failure) should be routinely screened for b-lactamase pro-
duction by one of the recommended tests, such as the Nitrocefin test.2 For the
Nitrocefin test, a dense suspension from several colonies is prepared in a small
tube with 0.2ml saline; 0.025ml of Nitrocefin is then added to the suspension
and mixed for one minute. A rapid change in the colour, from yellow to pink
or red, indicates that the strain produces b-lactamase.

Antimicrobial susceptibility testing of N. gonorrhoeae by the disc-diffusion
assay is not recommended in routine practice.

Genital specimens from women

The vaginal flora of premenopausal women normally consists predominantly
of lactobacilli, and of a wide variety of facultative aerobic and anaerobic
bacteria.

Abnormal vaginal discharge may be due to:

— vaginitis: Gardnerella vaginalis, Candida albicans;
— bacterial vaginosis: overgrowth of anaerobes and Mobiluncus spp.;
— cervicitis: Neisseria gonorrhoeae, Chlamydia trachomatis.

Other bacteria, such as Enterobacteriaceae, are not proven causes of vaginitis.
Vaginitis in prepubertal girls may be due to N. gonorrhoeae or C. trachomatis.

1 Van Dyck E, Meheus AZ, Piot P. Laboratory diagnosis of sexually transmitted diseases. Geneva,
World Health Organization, 1999.
2 The Nitrocefin reagent is obtainable from Oxoid Ltd, Wade Road, Basingstoke, Hants RG24
8PW, England, and consists of 1mg of Nitrocefin (SR112) and 1 vial of rehydration fluid (SR112A).
The tube test can be replaced by a disc test, using Nitrocefin-impregnated paper discs (Cefi-
nase discs, available from BD Diagnostic Systems, 7 Loveton Circle, Sparks, MD 21152, USA).

BLM1 1/17/04 2:02 PM Page 79

Page 88

Bacterial vaginosis (nonspecific vaginitis) is a condition characterized by an
excessive, malodorous, vaginal discharge associated with a significant
increase of Mobiluncus spp. and various obligate anaerobes, and a decrease in
the number of vaginal lactobacilli. A minimum diagnostic requirement for
bacterial vaginosis is the presence of at least three of the following signs:
abnormal vaginal discharge, vaginal pH > 4.5, clue cells (epithelial cells with
so many bacteria attached that the cell border becomes obscured), and a fishy,
amine-like odour when a drop of 10% potassium hydroxide is added to the
vaginal secretions.

Urethritis in women is also often caused by N. gonorrhoeae and C. trachomatis.

Ascending infections with N. gonorrhoeae, C. trachomatis, vaginal anaerobes,
and facultative anaerobic bacteria can cause pelvic inflammatory disease
(PID), with infertility or ectopic pregnancy as late sequelae.

Genital infections with bacterial agents, including N. gonorrhoeae and C.
trachomatis, during pregnancy may result in complications such as prema-
ture delivery, prolonged rupture of membranes, chorio-amnionitis, and post-
partum endometritis in the mother, and conjunctivitis, pneumonia, and
amniotic infection syndrome in the newborn.

On special request, cervicovaginal specimens may be cultured for bacterial
species, such as S. aureus (toxic shock syndrome), S. agalactiae (group B strep-
tococci, neonatal infection), Listeria monocytogenes (neonatal infection), and
Clostridium spp. (septic abortion).

Although infections with C. trachomatis and with human herpesvirus
are common, and can cause significant morbidity, their laboratory diag-
nosis requires expensive equipment and reagents and will not be discussed
here.

Collection and transport of specimens

All specimens should be collected during a pelvic examination using a specu-
lum. The speculum may be moistened with warm water before use, but anti-
septics or gynaecological exploration cream should not be used, since these
may be lethal to gonococci.

For examination for yeasts, G. vaginalis, and bacterial vaginosis, samples of
vaginal discharge may be obtained with a swab from the posterior fornix of
the vagina. Samples for gonococcal and chlamydial culture should be col-
lected in the endocervix. After inserting the speculum, cervical mucus should
be wiped off with a cotton wool ball. A sampling swab (see page 77) should
then be introduced into the cervical canal and rotated for at least 10 seconds
before withdrawal.

Urethral, anorectal, and oropharyngeal specimens for gonococci may be
obtained in a similar manner as from males.

In all cases of pelvic inflammatory disease (PID), as a minimum, the cervix
should be sampled for N. gonorrhoeae. Sampling from the fallopian tubes is
more reliable, but in most areas a cul-de-sac aspirate is the best sample
available.

In infants with ophthalmia neonatorum, conjunctival exudate should be col-
lected with a swab or a loop.

80

SEXUALLY TRANSMITTED DISEASES

BLM1 1/17/04 2:02 PM Page 80

Page 174

spp. 10, 15, 23, 61, 144
ASO test 135–137
culture preservation 16
surgical specimens 87, 91, 93, 150

Streptolysin O 135
Streptomycin 109
String test, stool specimens 49–50
Strips, diagnostic 151
Stuart transport medium 40, 62, 77, 81,

149, 151
Subcultures, blood samples 23
Subcutaneous tissue, infections 87
Sucrose fermentation 48, 51–53
Sulfamethoxazole 109
Sulfonamide 107, 108, 110, 116, 152
Sulfur granules 89
Supplements 151
Surgical specimens 86–87

collection/transport 89
culture 92–93
macroscopic evaluation 90–91
microscopic examination 91–92
pathogen identification 93–97
susceptibility testing 97

Surveys, quality assessment
programmes 17

Susceptibility testing 13–14, 103–121
anaerobic specimens 102
basic antimicrobials 107–109
benzylpenicillin 108, 110, 116
CSF cultures 29
direct vs indirect 117
general principles 103–105
indirect 117
Neisseria gonorrhoeae 79
plate size 118–119
recommended discs 152
sputum cultures 70–71
stool cultures 54
surgical specimens 97
throat specimens 65
treatment guide 106
urine cultures 36

Syphilis (see also Trepanoma pallidum) 76,
82, 83–85

serological tests 13, 125, 126–133

Tablets, diagnostic 151
Talampicillin 109
TCBS, see Thiosulfate citrate bile salts

sucrose agar
Tellurite agar 42, 48, 64, 148, 151
Tellurite discs 13, 23
Tellurite taurocholate gelatine agar

(TTGA) 42, 48
Temperature

equipment operating records 9
susceptibility tests 118

Terminology, microorganisms 5
Tests

agglutination 56–59, 125–126, 135–139
antimicrobial susceptibility 13–14,

103–121

166

INDEX

ASO 135–137
diffusion/dilution 103–104
febrile agglutinins 133–135
FTA-Abs 123, 124, 126, 127, 131–135
media performance 11–12
ONPG 13, 51, 146, 147, 151
PGUA 13, 35–36
rapid diagnostic 145, 148, 151
RPR 123–124, 126, 129–131
serological 12–13, 122–139
stool specimens 49–50

Tetanus 88, 98
Tetracycline 71, 107, 108, 110, 116
Thayer–Martin medium (MTM),

modified 12, 61, 78, 148, 149
Thiamphenicol 108
Thioglycollate broth 12, 23, 38, 92, 144,

150, 151
Thiosulfate citrate bile salts sucrose

(TCBS) agar 12, 42, 47–48, 147,
151

Throat swabs
collection 62
culture/identification 63–65
gonococcal pharyngitis 61

Thrombophlebitis 20
Tobramycin 107, 109, 110, 116
Tonsillitis 60, 61
Toxicity assays 38, 64
Toxigenicity 64
Toxic shock 20, 80
Transport (see also Dispatch)

anaerobic specimens 99
cervicovaginal specimens 81
CSF specimens 25
respiratory tract specimens 62
sputum specimens 68
STD specimen media 149
stool specimens 40, 146
surgical specimens 89
urethral specimens 77–78
urogenital specimens 81

Transport media 81
Amies 40, 41, 62, 66, 77, 81, 149, 151
Buffered glycerol saline 146, 152
Cary–Blair 40–41, 146, 151
Stuart 40, 62, 77, 81, 149, 151

Treatment, susceptibility tests as guide
106

Treponema
pallidum 76, 82, 83–85, 125–126, 149
vincentii 61

Tributyrin 70, 148–151
Trichomonas vaginalis 77, 81
Trimethoprim 78, 107, 110, 116, 152
Triple sugar iron agar 12, 70, 147
Trisodium phosphate 26, 73
Trypanosomiasis, African 26
Tryptic soy agar (TSA) 15, 63, 150
Tryptic soy broth (TSB) 22, 27, 96, 144,

150, 151
Tubercle bacillus, see Mycobacterium

tuberculosis

BLMINDEX 1/17/04 2:39 PM Page 166

Page 175

Tuberculosis
pulmonary 66, 67–68
VDRL test 127

Tuberculous meningitis 27–28
Turbidity standard

Kirby–Bauer technique 111–112
susceptibility testing 111

Typhoid fever 20, 37

Ulcerative pharyngitis, necrotizing
(Vincent angina) 61, 62

Ulcus molle, see Chancroid
Upper respiratory tract infections 60–65

carriers 62
causal agents 61–62
diagnostic reagents/media 148
direct microscopy 62
expected pathogens 147–148
pathogen culture/identification 63–65

Urea medium 12, 44–45, 76, 77
Ureaplasma urealyticum 77
Urease 12, 45, 47, 51, 53
Ureteritis 30
Urethral specimens

collection/transport 77–78
interpretation 78

Urethritis
in men 77–79
in women 80

Urinary tract infection (UTI) 30, 35, 94
Urine 30–36

culture/interpretation 32–35, 100
diagnostic reagents/media 145–146
expected pathogens 145
screening 32
specimen collection 30–32

Urogenital specimens 149
UTI, see Urinary tract infection

Vaginitis 76, 79, 81
Vaginosis 76, 79–82
Vancomycin 78, 110, 116, 149, 151, 152
VCN mixture 78, 151
VDRL, see Venereal Disease Research

Laboratory test
Venepuncture, blood collection 21
Venereal Disease Research Laboratory

(VDRL) test 123–124, 125
syphilis 126, 127–129

Ventricular puncture 25
V-factor 13, 144–151
Vibrio

cholerae 10, 16, 38, 146
agglutinating antisera 147, 153
identification 47–48, 52–53

167

INDEX

H

media 41, 42, 43
fluvialis 38, 53
furniss 53
hollisiae 38, 53
mimicus 38, 53
parahaemolyticus 38, 47–49
vibriostatic compound O:129 disc 147,

151
Vincent angina, see Necrotizing

ulcerative pharyngitis
Viral diarrhoea 41
Viral meningitis 27
Vitox 151
Voges–Prosgauer agar 11, 52, 53

Water-baths, quality control 8, 123
Weil–Felix reaction, rickettsias 133
White blood cells, see Leukocytes
Widal test, Salmonella typhi 6, 133
Wilkins–Chalgren anaerobe broth 100,

144, 151
Wounds

Clostridium spp. 98
nosocomial infections 88–90
penetrating 87–90, 96

Wright test, Brucella 133

XLD, see Xylose–lysine–deoxycholate
agar

XV-factor 13, 85, 144–145, 148–149, 151
Xylose–lysine–deoxycholate (XLD) agar

42–44, 48, 147

Yeasts (see also Fungi) 26, 69
Yersinia

enterocolitica 17, 39, 133, 146
colony morphology 48
identification 47, 52–53, 59
media 41, 42, 43

frederiksenii 53
intermedia 53
kristensenii 53
pseudotuberculosis 53

Zephiran–trisodium phosphate
procedure 73–74

Ziehl–Neelsen staining 74, 152
CSF specimens 27, 28
diagnostic specificity 6
pulmonary tuberculosis 67
sputum specimens 67
surgical specimens 92, 93

Zone diameters, disc-diffusion
susceptibility testing 104–105,
110–111, 116–119

BLMINDEX 1/17/04 2:39 PM Page 167

Similer Documents