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www.elsevier.es/eimc

Continuing medical education: Methods of rapid diagnosis

Rapid diagnosis of sexually transmitted infections夽

Luis Otero-Guerra a , Ana Fernández-Blázquez b , Fernando Vazquez b,c,d,∗
a
Servicio de Microbiología, Hospital de Cabueñes, Gijón, Spain
b
Servicio de Microbiología, Hospital Universitario Central de Asturias, Oviedo, Spain
c
Departamento de Biología Funcional, Área de Microbiología, Facultad de Medicina, Oviedo, Spain
d
Fundación de Investigación Oftalmológica, Instituto Oftalmológico Fernández-Vega, Oviedo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Sexually transmitted infections (STIs) are responsible for an enormous burden of morbidity and mortality.
Received 3 January 2017 Worldwide, millions of cases of STIs, such as syphilis, chlamydia, or gonorrhoea occur every year, and
Accepted 5 January 2017 there is now an increase in antimicrobial resistance in pathogens, such as gonococcus. Delay in diagnosis
Available online xxx
is one of the factors that justifies the difficulty in controlling these infections. Rapid diagnostic tests allow
the introduction of aetiological treatment at the first visit, and also leads to treating symptomatic and
Keywords: asymptomatic patients more effectively, as well as to interrupt the epidemiological transmission chain
Point of care system
without delay. The World Health Organisation includes these tests in its global strategy against STIs.
Screening assay
Sexually transmitted diseases © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiologı́a
Clı́nica. All rights reserved.

Diagnóstico rápido de las infecciones de transmisión sexual

r e s u m e n

Palabras clave: Las infecciones de transmisión sexual (ITS) suponen una importante carga de morbimortalidad. A nivel
Sistemas de diagnóstico a la cabecera del mundial todos los años se producen millones de casos de ITS como sífilis, infección por clamidias o gono-
enfermo cocia, y actualmente se asiste a un incremento de la resistencia a los antimicrobianos en patógenos como
Cribado de ITS
el gonococo. La demora en el diagnóstico es uno de los factores que justifica la dificultad para controlar
Infecciones de transmisión sexual
estas infecciones. Las pruebas de diagnóstico rápido permiten instaurar el tratamiento etiológico en la
primera consulta, lo que lleva a tratar a más pacientes, tanto sintomáticos como asintomáticos, de forma
más efectiva, e interrumpir sin demoras la cadena epidemiológica de transmisión. La OMS incluye estas
pruebas en su estrategia mundial contra las ITS.
© 2017 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiologı́a
Clı́nica. Todos los derechos reservados.

Introduction seems far-removed from developed countries. Syphilis causes over

300,000 foetal and neonatal deaths per year and exposes another
Worldwide, sexually transmitted infections (STIs) pose a signif- 215,000 children to premature death. It also estimates that 357
icant morbidity and mortality burden as they compromise quality million new cases of four types of curable STIs are recorded every
of life, sexual and reproductive health and newborn and child year: 131 million Chlamydia trachomatis (CT) infections, 78 million
health. They also indirectly facilitate the transmission of the human Neisseria gonorrhoeae (NG) infections, 6 million Treponema pallidum
immunodeficiency virus (HIV) and cause cell changes that precede (TP) infections and 142 million Trichomonas vaginalis (TV) infec-
some forms of cancer. The WHO provides data on a reality that tions. Similarly, the human papillomavirus (HPV) is believed to
be responsible for 530,000 cases of cervical and uterine cancer
as well as 264,000 deaths. We also cannot forget the emergence
DOI of original article: http://dx.doi.org/10.1016/j.eimc.2017.01.004 of multidrug-resistant gonococcal strains that threaten us with
夽 Please cite this article as: Otero-Guerra L, Fernández-Blázquez A, Vazquez F. untreatable gonorrhoea.
Diagnóstico rápido de las infecciones de transmisión sexual. Enferm Infecc Microbiol While it is true that progress has been made, such as the
Clin. 2017. http://dx.doi.org/10.1016/j.eimc.2017.01.004
∗ Corresponding author.
reduction of mother-to-child syphilis transmission in developing
E-mail address: opsklins@gmail.com (F. Vazquez).
countries, globally the prevalence of STIs remains the same or is

2529-993X/© 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiologı́a Clı́nica. All rights reserved.

EIMCE-1658; No. of Pages 7

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Table 1 POCTs must also comply with quality controls included in the
Differences between rapid tests and POCTs.
tests and be environmentally safe and low cost.
Characteristic Rapid testing POCTs An important aspect is what users think the qualities of rapid
Difficulty Simple Simple (can be used in tests/POCTs should be in STIs. In a study on focus groups, it was
primary care) found that the qualities of a rapid test should be high sensitivity
Time <30 min <20–30 min and specificity, a short procedure time and a low cost.5
Electricity source Yes, necessary No For POCTs, various types of technology are used6 :
Trained personnel Yes, necessary No
Quality control No, internal controls Yes (more reliable)

POCTs: point-of-care tests. a) Precipitation/agglutination reactions such as the RPR test for
Source: Adapted from Muralidhar.3 syphilis.
b) Immunochromatography in different test formats: (b1) lateral
flow; (b2) multiple, e.g. HIV + syphilis, HIV + syphilis + HBV/HCV,
treponemal test + non-treponemal test; (b3) using a flow assay
even on the rise. Numerous factors contribute to this, but the treat-
such as dot-blot; (b4) with readers/scanners to eliminate
ment delay that arises while patients await a diagnostic result
observer bias, thereby increasing sensitivity and facilitating
is particularly important. By means of a mathematical model, it
quantification.
has been shown that using a rapid diagnostic test with a sensitiv-
c) Emerging technologies such as microfluidic assays (which detect
ity as low as 63% successfully improves the percentage of treated
multiple analytes such as HIV and syphilis) and loop-mediated
patients, compared to waiting for the result of a high-sensitivity
isothermal amplification technology (LAMP), which achieves
test available at a second consultation, which many patients fail to
amplification using four primers and polymerase enzyme in a
attend.1 As such, diagnostic tests that provide immediate results
constant temperature reaction (60–65 ◦ C) and obtains results in
will facilitate the administration of aetiological treatments to a
less than 1 h.
larger number of infected patients and new transmissions will be
avoided by breaking the chain of infection.
To that effect, it is necessary to reinforce the capacity of the POCTs were developed to complement centralisation in “core”
laboratories and to also proceed with designing and implement- laboratories, allowing for decentralised determinations that are
ing diagnostic tests at the point of care to facilitate systematic and available 24 h a day, 365 days a year. They are usually combina-
early STI diagnosis of all suspected individuals, even if they are tions of syndrome-based microorganisms, can be collected by the
asymptomatic. patients themselves, do not require trained personnel and are rapid
In this review, we will present the latest rapid diagnostic tech- so as to enable decision-making. Of all of them, the STI tests are
niques for the main STI- and vulvovaginitis-causing pathogens, among those that have been proven cost-effective.7
though we will leave viral hepatitis and HIV to one side. Irrespective of the terminology and discourse that may surround
rapid tests or POCTs, in this review we include the types of tests we
consider to be interchangeable, whether these are with or without
History, concept and characteristics of the rapid tests in equipment at the point of care or in a nearby laboratory, and which
sexually transmitted infections try to provide the fastest results possible.

In STIs, diagnostic tests can serve several purposes: (a) diagno-

sis; (b) screening of high-risk groups; (c) treatment monitoring; (d) The rapid response laboratory in sexually transmitted
epidemiological surveillance; (e) investigation of outbreaks; (f) val- infections and needs in different settings: primary care,
idation of syndrome management in countries with few resources; emergency departments, STI clinics, reference laboratories
(g) detection of resistance patterns; (h) ensuring quality in labora-
tory tests; and (i) research.2 Laboratory centralisation makes the presence of a rapid
The diagnostic tests used in STIs are: (a) direct microscopy; response laboratory difficult in our setting, but the following meth-
(b) culture; (c) antigen detection; (d) serology; (e) detection of ods may be available in various working environments:
microbial metabolites (whiff test, for example), and (f) molecular
methods. All of these may be considered rapid at least in some of a) Primary care: e.g. Gram stain and Amsel criteria for bacterial
their forms, except culture. vaginosis.
Rapid tests in STIs can be considered as such, or as point-of-care b) Emergency departments: normally have a support laboratory.
tests (POCTs). This review considers both types indiscriminately. c) STI clinic: the above methods, dark-field microscopy, urine sed-
POCTs can be defined as diagnostic tests that allow a diagnosis iment.
to be obtained and a treatment indicated at the same visit. The d) Reference laboratory: all rapid techniques.
differences between them are detailed in Table 1 (adapted from
Muralidhar3 ). In developing countries, rapid tests/POCTs generate huge inter-
Rapid tests/POCTs aimed at STI diagnosis must meet the follow- est as they help to reduce the enormous disease burden that they
ing requirements established by the WHO4 : endure. These tests allow a move away from syndromic treat-
ASSURED ments (targeted blindly and simultaneously at various pathogens
causing the same syndrome) and a progression towards aetiolog-
• Affordable. ical treatments (targeted specifically at the causal pathogen). The
• Sensitive. unnecessary use of antimicrobials and the emergence of resistances
• Specific. are avoided, costs are reduced and the chain of infection is broken
• User-friendly (few steps and minimum training). more effectively. However, there are drawbacks, as aspects such
• Rapid and robust (storable at room temperature, results in as cost, the need for the refrigeration of reagents or electrical sup-
<30 min). ply requirements (e.g. for a microscope) can mean that tests which
• Equipment-free. prove very useful in one setting may be useless in another. As such,
• Deliverable to end-users. all of the ASSURED requirements are equally important.
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Table 2
List of rapid tests for sexually transmitted infections.

Microorganism/syndrome Test Se (%) Sp (%) Comments

Chlamydia trachomatis Giemsa stain, Macchiavello-Gimenez stain Low Low

Immunochromatography 82–84 98
GeneXpert CT/NG NAAT 97–99 >99
Neisseria gonorrhoeae Endocervical Gram stain 45–65 90–99 Pharyngeal and rectal Gram stains are not
recommended
Urethral Gram stain (with symptoms) 90–95 95–99
Urethral Gram stain (asymptomatic) 50–75 85–87
GeneXpert CT/NG NAAT 98–100 99.9
Treponema pallidum Dark-field microscopy 80–90; 39–81 <100; 82–100 Not for oral or rectal lesions
Direct immunofluorescence 90–95 >98 Requires a fluorescence microscope and
trained personnel
Herpes simplex RPR (non-treponemal antibodies) 73–100 79–98 Requires confirmation
Tzanck smear Variable No Sp Depends on the no. of viral particles in lesions
70–90% Se if sufficient no. of cells
Haemophilus ducreyi Direct immunofluorescence 70–90 >95
Donovanosis Gram stain <50 50–70
Bacterial vaginosis Wright-Giemsa stain 40–50 <50
Wet mount 70–90 95–100
Gram stain 60–80 95–100
pH 75–80 60–70
DNA probe (Affirm VPIII) >90 >99
Rapid pH and Rapid Amine (FermCard) 80–90 85–90
Rapid PIP (G. vaginalis) 80–85 90–92
Candida spp. BVBlue system 91.7 97.8
Wet mount 40–60 >99
Examination with KOH 10% 54–80 96
Trichomonas vaginalis DNA probe (Affirm VPIII) 85–90 >99
Wet mount 62–92 99–100 70% compared to PCR
Latex agglutination 95 100
OSOM Trichomonas Rapid Test 80–94 95

Se: sensitivity; Sp: specificity.

Source: Adapted from Aznar Martín et al.8

Rapid diagnostic tests in sexually transmitted infections Chancroid

and their evidence
At present, the use of the PCR technique is indicated for the
Table 2 shows the rapid techniques for STIs (adapted from Aznar detection of Haemophilus ducreyi (HD), due to the low sensitivity of
Martín et al.8 ). Gram staining (typical in “schools of fish” with small pleomorphic
Gram-negative bacilli). In comparison to culture, microscopy has a
sensitivity of 50%, and false positives are also common.11 A multi-
Candidiasis format PCR is available on the market for HD and TP.

Microscopy and culture are the tests that should routinely be

performed in symptomatic women (level of evidence III, grade B).9 Chlamydia
The microscopic observation of yeast in vaginal discharge by means
of a wet mount or Gram stain examination has the advantage of The majority of the diagnostic tests available are carried out in
being rapid, but has a low sensitivity (50%). the laboratory and there may be a delay between collection and the
Criteria for diagnosing vaginal candidiasis (level of evidence III, result.
grade B)10 : The urethral Gram stain is the standard diagnostic test for non-
gonococcal urethritis, but is dependent on the observer and has a
low specificity. As such, the use of flow cytometry is also currently
- Absence of fishy odour in the “whiff test”, using a potassium
being introduced to detect leukocyturia in urine as an inflamma-
hydroxide solution on a speculum, and amine odour test on a
tory marker of nongonococcal urethritis (NGU) caused by CT and
slide, are supportive, since candidiasis does not usually coexist
Mycoplasma genitalium (MG).12
with bacterial vaginosis or TV infection, but they are not diagnos-
EIA-based POCTs have a low sensitivity, although new forms
tic.
present sensitivities of 82–84% compared to nucleic acid amplifi-
- Presence of yeasts or pseudohyphae on the wet mount examina-
cation tests (NAATs).13,14
tion of vaginal discharge (40–60% sensitivity).
NAATs are the diagnostic benchmark but are generally car-
- Presence of yeasts or pseudohyphae on the Gram stain of vaginal
ried out in the laboratory, taking several hours. New formats like
discharge (up to 65% sensitivity).
isothermal NAATs (LAMP) are commercially available, offer good
sensitivity (97.1%) and specificity (97.9%), and have an execution
The BD Affirm VPIIITM system (Becton Dickinson, Franklin Lakes, time of less than 1 h. However, performing the techniques is com-
New Jersey, United States) uses hybridisation to determine the plex and should be done by trained personnel; they therefore
presence of the genetic material of TV, Gardnerella vaginalis (GV) cannot be used as POCTs.
and certain Candida species (Candida albicans, Candida glabrata, New NAAT-based techniques (GeneXpert CT/NG, Cepheid, Sun-
Candida kefyr, Candida krusei, Candida parapsilosis and Candida trop- nyvale, CA, United States) are appropriate for genital samples and
icalis), with a Candida sensitivity of 85–90% and a specificity of >99%. reduce the time required to issue a result.15 Extra-genital sam-
Results are obtained in less than 1 h. ples require additional validations.16 Their sensitivity is very high
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Table 3
Sensitivity of the Gram stain with ≥5 PMNs for Chlamydia trachomatis and Ureaplasma urealyticum.

Microorganism Janier et al., 199524 Iwuji et al., 200825 Orellana et al., 201223

Chlamydia trachomatis 29% 73% 23%

Ureaplasma urealyticum 33% – 11%

PMN: polymorphonuclear leukocytes.

(97–99%) as is their specificity (>99%), for endocervical, vaginal and Table 4

Sensitivity and specificity according to PMN threshold.
urine samples.17
No. of PMNs Se (%) Sp (%)
Lymphogranuloma venereum (LGV) >2 38 79

≥5 26 91
Diagnosis requires clinical suspicion and very specialised lab- Neisseria gonorrhoeae 80
oratory tests are necessary for confirmation. Firstly, the presence Chlamydia trachomatis 23
of CT must be seen in the lesions, usually using NAATs. The sam- Ureaplasma urealyticum 11
ples used are both genital and extra-genital: rectal, pharyngeal and PMN: polymorphonuclear leukocytes; Se: sensitivity; Sp: specificity.
urine.18 Chlamydia-positive samples should be assessed a second
time using a specific PCR reaction for LGV-producing chlamydia
DNA. Typing techniques are also used, by means of RFLP or sequenc- determine the existence of urethritis, a cut-off point of ≥5 PMN
ing. In light of the above, no rapid detection method exists at leukocytes is not very sensitive (Table 3), and that lowering it to
present, although the GeneXpert PCR could meet these criteria. ≥2 PMN leukocytes increases sensitivity (Table 4).
However, this is usually used in a second step, when the presence b) Methylene blue stain for urethritis: another aspect to take into
of chlamydia has already been detected. account in stains is whether the Gram stain is superior to oth-
ers. The Gram stain has been compared to the methylene blue
Gonorrhoea stain and a mixture of methylene blue and crystal violet (4:1
parts),27,28 which favours the intra or extracellular examination
Gonorrhoea diagnosis is established with the detection of NG of diplococci.
at the infection site. The method to be used will depend on the The main advantage is that it takes less time to perform: 4 min
patient’s symptoms, transport conditions to the laboratory and the (Gram) versus 10–15 s (methylene blue), with a sensitivity of
available tests. 97.3%, a specificity of 99.6% and a concordance of 100%. Although
the bacterial morphology cannot be seen with its stain charac-
a) Gram stain for urethritis: Gram staining is a rapid technique teristics in methylene blue, this is not important for examining
that is equally as sensitive as culture in symptomatic urethri- the presence of PMN leukocytes and, thus, urethritis.
tis among men, though it has limited sensitivity in other areas. c) Gram stain for cervicitis: this is performed on cervical discharge
Examinations should be performed with a 1000× objective lens and >10 PMN leukocytes with a 1000× objective lens is deemed
under oil immersion for at least 2 min, checking for the presence to be suggestive of infection.
of polymorphonuclear (PMN) leukocytes, a pink nucleus and a d) NAATs: NG detection with NAATs is more sensitive than culture
colourless cytoplasm, usually >4–5 PMN leukocytes per immer- and may be performed on a wide range of samples: urethral
sion field. If a urinary Gram stain is performed, it is advisable or urine in men, and endocervical or vaginal in women. Urine
to collect the first 10–15 ml from the first part of urination as samples in women have a lower sensitivity and they are not an
well as to observe the sediment after centrifugation to check optimal sample (II, B).29
for the presence of ≥10 PMN leukocytes. Gonorrhoea appears as In the rectum and pharynx, NAATs are more sensitive than
kidney-shaped, oval, Gram-negative cocci, in intra and extracel- culture, but positive samples should be confirmed with another
lular pairs.8 In urethral samples from symptomatic men, Gram NAAT that uses a different target (III, C).
staining offers a high sensitivity (90–95%) and facilitates imme- In any case, in their current formats, NAATs cannot be con-
diate diagnosis (III, C).19 The sensitivity of the same samples in sidered rapid tests, although the incorporation of the LAMP
asymptomatic men is distinctly lower (50–75%). Gram staining technique renders it adequate in terms of its rapidness, sensitiv-
of rectal samples is not recommended in symptomatic patients ity and specificity. A LAMP technique by the same manufacturer
or in urethral samples from women (III, C). It should also not be is available on the market, with an identical format and char-
used on pharyngeal samples. acteristics to the one mentioned previously for chlamydia.
The traditional criterion for determining the existence of However, although rapid (<1 h), it cannot be used as a POCT due
urethritis is the observation of 4–5 PMN leukocytes with an to its complexity.
immersion field objective lens and the presence or absence of The GeneXpert CT/NG NAAT, with the characteristics cited
Gram-negative diplococci, in both gonococcal or nongonococ- in the “Chlamydia” section, presents excellent sensitivity
cal urethritis.20 This criterion was based on the best sensitivity (98–100%) and specificity (99.9%) in vaginal and endocervical
and specificity established with NG and CT cultures. However, samples as well as male urine, with lower results in urine sam-
with NAAT systems, it has been seen to be too strict for NGU, ples from women.17
also considering variations between microscopists and the fact
that sensitivity varies according to the grade of urethritis (low- Syphilis
grade cases present more false negatives). Considering there
to be no urethritis with the presence of <5 PMN leukocytes Rapid tests for diagnosing syphilis through the detection of anti-
causes 32% of CT-induced cases to go undetected, along with bodies have shown excellent results in prenatal syphilis screening,
37% caused by MG, 38% by adenovirus and 44% by herpes virus; particularly in developing countries. Opportunities for accessing
it is thus deduced that this is not a good cut-off point for ruling pregnant women are rare, and diagnosis—and, where applicable,
out infection.21 Various studies22–26 highlight that, in order to treatment—have to be given at the time of the consultation. It is
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estimated that in Sub-Saharan Africa, where there is a syphilis sero- case with a sensitivity of 89.2% and a specificity of 99.3% compared
prevalence of 8.3%, only 38% of pregnant women have access to to culture.
neonatal screening, so half a million children are believed to die Moreover, there are POCTs such as the OSOM Trichomonas Rapid
every year due to congenital syphilis sequelae.6 With regard to Test (Sekisui Diagnostic, Hartwell Place, Lexington, United States),
the serological laboratory techniques used in developed countries which determines the presence of the antigen and has shown a
that require a centrifuge, new immunochromatography techniques high sensitivity (80–94%) and specificity (95%). No instruments are
may be considered authentic POCTs given that they can be per- required and it provides results in 30 min, although it must be taken
formed on whole blood obtained by finger prick, the reagents do into account that false positives are possible (IIb, B).32
not require refrigeration and they obtain results in 15–20 min.
Although they are easy to perform, the manufacturer’s instructions
must be strictly adhered to in order to guarantee accuracy. These Herpes simplex virus (HSV)
techniques use recombinant antigens and capture specific trepone-
mal antibodies, so the exclusive performance of this test leads to There are various rapid techniques:
unnecessary treatments in previously treated and cured patients
who continue to present lifelong treponemal antibodies. In such
cases that come back positive during the initial screening period, a) Optical microscopy. This technique is inadequate. Direct exam-
referrals to reference laboratories would be ideal in order to deter- ination using the Tzanck or Papanicolaou smear has a limited
mine the disease activity with non-treponemal tests. A dual POCT sensitivity and is nonspecific. It does not differentiate between
system that separately determines the presence of treponemal and HSV-1 and 2, and Cowdry type A intranuclear inclusions are not
non-treponemal antibodies is available on the market, offering exclusive to the HSV. Moreover, a negative result does not rule
diagnostic confirmation when both are positive. However, the lack out the possibility of genital herpes.
of non-treponemal antibody quantification inhibits the determina- b) Electron microscopy. This has fallen out of use as it requires
tion of the initial titre for treatment control. Dual determination is concentrations of over 108 virions/ml. It provides rapid results
also possible in the same antibody assay for syphilis and HIV. but has a limited sensitivity and requires equipment that is not
Things are very different in our setting, and serological tests are widely available in clinical laboratories.
usually carried out in the context of other laboratory tests. An ini- c) Direct immunofluorescence. HSV antigens can be detected with
tial positive non-treponemal test result would be confirmed with immunofluorescence or immunoenzyme techniques. These are
another treponemal test, and the same is true in reverse; if we carry rapid techniques with a sensitivity and specificity ranging from
out an initial treponemal test (usually an EIA), we confirm a posi- 70% to 90% in symptomatic patients. On direct samples, sen-
tive result with a non-treponemal test (reverse algorithm). These sitivity reduces as the lesion develops over time. The use of
tests are performed in a scheduled manner in the laboratory. monoclonal antibodies provides good specificity and facilitates
However, it is clear that in certain situations where an immedi- the differentiation of HSV-1 and 2. It is currently used more as a
ate diagnosis prevails over other considerations, the performance of complement to identify viruses in cell cultures.
an RPR test only requires one centrifuge, the reagent and the person d) NAATs are recommended for the diagnosis of genital herpes (Ib,
responsible for performing the technique having the knowledge to A).33 Detecting HSV DNA using PCR techniques increases detec-
do so. tion by 11–71% compared to culture. It also allows for more
Dark-field microscopy requires a microscope with the afore- lenient conditions than culture with regard to sample storage
mentioned characteristics (which not all laboratories have) and a and transportation. It allows for the differentiation of HSV-1
microscopist who is an expert in the technique, which is consid- and 2, which should be established in all new diagnoses (III, B).
erably complex for the observer. Non-genital samples, especially Positive results do not require confirmation.
oral samples, may contain nonpathogenic treponemes that lead
to false positives, so their use is not recommended. There is a IIA
recommendation for performing dark-field microscopy on chancre Vaginosis
samples, provided an expert microscopist and adequate equipment
are available.30 Vaginosis may be diagnosed on the basis of three sets of criteria
PCR techniques, on the other hand, may be used on (Amsel, Nugent or Ison) (II, B).10
oral and other extra-genital and genital samples, with a IA The Amsel clinical criteria34 are based on the presence of three
recommendation,30 since commensal treponemes do not interfere of the following signs or symptoms: (a) thin, homogeneous, white,
with the technique. These techniques are only available at referral uniform vaginal discharge that adheres to the vaginal walls; (b)
centres, but are not considered “rapid response” unless they are of vaginal pH >4.5; (c) fishy odour after adding 10% KOH to the sam-
the LAMP variety. ple; and (d) more than 20% clue cells in wet mount preparations
(microscope with a 40× objective lens).
Trichomoniasis It may be confirmed by objective microscopic criteria viewed in
a Gram stain of vaginal discharge using the Nugent35 score (Table 5).
A wet mount examination is easy to perform, rapid, low-cost This is considered the gold standard. The microbiological diagnosis
and highly specific (98%), but has a low sensitivity (62–92%) and is of bacterial vaginosis is carried out using a Gram stain of vagi-
dependent on the observer. To perform this test, a drop of urethral nal discharge, determining the relative quantity of morphotypes
or vaginal discharge is mixed on a slide with a drop of 0.5% phys- characteristic of abnormal vaginal microbiota (Gram-positive and
iological saline solution at 37 ◦ C. A coverslip is then applied and Gram-negative bacilli and curved bacteria) and the presence of
it is observed under a microscope to check for the characteristic clue cells (epithelial cells coated with Gram-positive and Gram-
movements of trichomonas. The preparation should be examined negative morphotypes with obscured borders). Stained cervical
within 10 min of taking the sample, as the characteristic movement smears using the Papanicolaou method are not appropriate due to
of the trichomonas parasite reduces after this time, thus hindering their low sensitivity.
its identification.31 However, the BASHH guidelines recommend (grade C) the use
As mentioned previously in the “Candidiasis” section, the BD of the Ison and Hay criteria,36 as they reflect real microbiota possi-
Affirm VPIIITM system also determines the presence of TV, in this bilities better than the Nugent criteria.
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Table 5
Interpretation of the Nugent criteria.

Score Gram-positive bacilli (Lactobacillus spp.) Small Gram-negative bacilli (Gardnerella vaginalis) Curved Gram-negative bacilli (Mobiluncus spp.)

0 4+ (>30 morphotypes/field) 0 0
1 3+ (5–30 morphotypes/field) 1+ (<1 morphotype/field) 1+ 2+ (<5 morphotypes/field)
2 2+ (1–4 morphotypes/field) 2+ (1–4 morphotypes/field) 3+ 4+ (>5 morphotypes/field)
3 1+ (<1 morphotype/field) 3+ (5–30 morphotypes/field) –
4 0 4+ (>30 morphotypes/field) –

Grade 0: Unrelated to bacterial vaginosis; epithelial cells only, with We have rapid tests for syphilis and HIV but still lack genuinely
no Lactobacillus morphotypes. May indicate recent antibiotic use. rapid point-of-care tests that enable the diagnosis of highly preva-
Grade 1 (normal): Lactobacillus morphotypes predominate. lent and curable infections, such as those caused by CT, TV and NG,
Grade 2 (intermediate): mixed flora with some lactobacilli present, within a matter of minutes. Generally speaking, the available meth-
but Gardnerella or Mobiluncus morphotypes also present. ods do not reach the sensitivity and specificity levels attained by
Grade 3 (bacterial vaginosis): predominantly Gardnerella and/or PCR techniques.
Mobiluncus morphotypes. Few or absent lactobacilli. Among the most promising tests described in the litera-
Grade 4: unrelated to bacterial vaginosis; only Gram-positive cocci ture, we can site the assay for CT with microwave accelerated
observed, no lactobacilli. metal enhanced fluorescence (MAMEF) technology, which provides
results in 9 min with a sensitivity of 82% and a specificity of 93%.39
Commercial systems also exist, although some are not marketed Moreover, hopes are high regarding microfluidic techniques,
in Spain: which are microsystems that incorporate assay operations and
sample preparation on a chip, with the advantages being that they
OSOM BVBlue (Sekisui Diagnostic, Hartwell Place, Lexington, require very small quantities of sample, have a faster response time
United States) measures sialidase levels. and are portable. Generally speaking, these techniques work well
Pip Activity TestCard (Litmus Concepts Inc, Santa Clara, California, for antibody detection due to the abundance of target molecules.
United States) assesses proline aminopeptidase. For other samples, such as urine or those obtained with a swab,
BD Affirm VPIII (Becton Dickinson, Franklin Lakes, New Jersey, the sample will require prior processing before it is added to the
United States) uses a DNA probe to detect high concentrations of microsystem. This presents limitations for antigen detection due to
GV, with a sensitivity of over 90% and a specificity of over 99%. few target molecules being found in the sample.
PCR-based systems have also been described but are not available The future also lies in determining the presence of antimicrobial
on the market. resistances, which is especially important for NG and MG. Work on
array and microfluidic formats is currently only being conducted at
Point-of-care tests (POCTs) and multiple platforms highly-specialised laboratories, although it would be ideal if they
could be used at the point of care.
In relation to POCTs, the most widely implemented tests at
present are for HIV, followed by TP. As for gonorrhoea, the same Conflicts of interest
problems regarding cross-reactions continue to arise, and there is
limited experience with others, such as those used for the HPV or None.
HSV.
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