Only if C. trachomatis NAATs are not available or affordable, isolation of C. trachomatis in cell culture or identification of C trachomatis by direct fluorescence assays (DFA) can be used for diagnosis of acute infections. Validated and quality assured NAATs are recommended due to their superior sensitivity, specificity and speed of diagnosis of both symptomatic and asymptomatic chamydial infections compared to all other diagnostic techniques.15,16 Due to the high specificity of the appropriately validated C trachomatis NAATs and risk of loasing low positive result in repetead testing, confirmatory testing of positive specimens is not recommended.17,18 An important exception is represented by legal investigations in case of sexual assault.

The NAATs can use less invasively colleted specimens such as urine samples in men or vulvo-vaginal swabs in women and anorectal swabs in both genders.19 Diagnostic sensitivity can be increased using coated magnetic beads nucleic acids nucleic acids were isolated in higher quantity and quality.20 These bead based extraction systems can be automated and use in several high-throughput systems that allow simultaneous testing of chlamydia and gonococci with sensitivity and specificity.20 In order to increase the sensitivity of the technique, these tests are based on the detection of genes present in high number of copies (cryptic plasmid X06707 (10 copies /genome) or 16S ADNr (2 copies/genome).21

NAATs have detected 10-30% more C. trachomatis positive specimens than culture in studies comparing the two methods.22,23 In some several studies, results of different NAATs were show to be highly concordant.24,25 The importance of genetic variation became evident with the appearance of the Swedish variant that was not detected by some commercial NAATs due to a deletion in the target region of these test.26 The implementation of a 2nd target region in NAATs represents an important improvement of NAATs, allowing detection of new variants with deletions or recombination in one of the target region.27 As an example of systems that developed a modification of the technique Cobas Taqman CT/NG v 2.0 (Roche) and Artus C. trachomatis plus RG Kit PCR (Qiagen). One limitation of all these diagnostic techniques is the lack of discrimination between the different biovars of C. trachomatis related to different pathological processes that can be detected in urethral or cervical samples. None of the above technique can discriminate between serovars D-K and serovars L1-L3 related with lymphogranuloma venereum (LGV).21

Rapid point-of-care test provide a quick and easy test result, and diagnosis and subsequent treatment can be provided at the same visit at clinic or even in remote setting. Most POCTs are immune chromatographic test based on lateral flow technology and detec chamydia lipopolysaccharide antigen (LPS) in genital swabs or urine. Compared with culture and NAATs, these antigen based in POCTs are significantly less sensitive and less specific. The antigen based POCTs were not recommended for C. trachomatis testing of both asymptomatic screening and symptomatic patients.28 POCTs with increased sensitivity have been developed and new POC NAATs (e.g. Xpert assay of Cepheid CT/NG). This assay is based on real time PCR carried out in a closed system. After application of the clinical sample to a cartridge, the subsequent steps of nucleic acid isolation, amplication and detection of PCR products proceed in a fully automated process. Another commercial test POCTs NAATs use technology isothermal amplification, like loop mediated isothermal ampllication (LAMP) or recombinase polymerase amplification (RPA).29

Testing for C. trachomatis antibodies is not useful to diagnose local epithelial infectious of the lower genital tract, because antibodies are detectable with a delay of several weeks, antibody titers may be low, and many serologic test are not able to differentiate antibodies against different chlamydia species.

The microinmmunefluorescence (MIF) test was long time considered the reference method of chamydia antibody testing but enzyme immunoassays (EIA) and inmunoblosts or line assays are currently used more frequently to detect clamydia infections.30,31 The EIA technology is based on the detection of antigen by measuring a colored signal generated by the antigen reaction liposaccharides (LPS) with the antibody. Traditionally they have enjoyed great popularity for being simple, objective and automated techniques. The specificity of EIA is low, being able to give false positives due to the presence of bacterial lipopolisaccharides LPS. Other techniques are based on direct staining of samples with fluorescein-labeled monoclonal antibodies (DFA). This last technique uses species-specific antibodies directed mainly against the antigen major outer membrane protein (MOMP) and to a lesser extent against the LPS. The main advantages of the DFA techniques are its speed (30 min) and specificity close to 100%, the sensitivity is 85-90%, compared to the culture and does not require specific means of transport. Among its drawbacks is the subjective interpretation and requires experienced staff, low reproducibility and the volume of samples should not be high.

Until the end of the 20th century, cell culture has been the reference standard against which all other tests have been compared. But mainly due to the appearance of new diagnostic methods easier to implement, fast and sensitive, cell culture has been relegated to reference laboratories. Established cell lines for isolation of C. trachomatis include McCoy, Hela 29.21 The specimens for culture must be collected using special devices and transport media. Sensitivity of culture may be impaired by inadequate specimen collection, storage and transport, toxic substances in clinical specimens and overgrowth of cell cultures by commensal bacteria and fungi. Cell culture is a very specific technique, however sensitivity is not very good 75-80%.21,32

NG could be visualized on microscopy of a stained genital tract smear in symptomatic patients. In men with urethral discharge, microscopy (x1000) using Gram for identification of diplococci within polymorphonuclear leukocytes has good sensitivity (≥95%) and specificity (≥99%) as a rapid diagnostic test.36 However, in asymptomatic men, this technique has poor sensitivity (≤55%), as well as in identifying endocervical or rectal infection (≤55% and ≤40%, respectively); in these circumstances, microscopy cannot be recommended as a test for ruling out infection. In addition, Gram stains of endocervical, rectal, or pharyngeal specimens also are not recommended to detect infection due to poor specificity as well as low sensitivity.

Culture is the only diagnostic test that allows antimicrobial susceptibility testing, remaining important to detect and monitor antimicrobial resistance. Specimens should be obtained by using swabs other than those composed by wood and cotton because they may be inhibitory or toxic to NG. Some transport systems can maintain gonococcal viability for up to 48 h in ambient temperature.37 Swabs should be inserted 2-3 cm in the male urethra or 1-2 cm into the endocervical canal followed by 2-3 rotations.

Specimens from sterile sites could be cultured on nonselective medium (e.g. chocolate agar), whereas those from nonsterile locations are cultured on a selective mediun (e.g. Martin-Lewis, Thayer-Martin),38 which contain antimicrobial agents that inhibit the growth of other bacteria and fungi. These media are incubated at 35 °C in an atmosphere supplemented with 5% CO2 and examined during at least 48-72 h. Gram-negative diplococci and oxidase-positive colonies can be presumptively identified as NG. Further additional biochemical tests are needed in order to confirm the diagnosis.

Culture should be carry out for antimicrobial sensitivity study in patients with persisting infection or if treatment failure is suspected. Moreover, characterization of isolates by molecular typing may be a useful tool to predict antimicrobial resistance due to the fact that some types are associated to decreased susceptibility to several antibiotics.39 The sensitivity of culture is high for genital samples but strongly depend on the specimen collection, transport, storage and isolation procedures.

NAATs techniques are recommended for detection of infections caused by NG with and without symptoms. NAATs are more sensitive than culture, they can be used on a wider range of specimen types, and specimen quality, transportation and storage are less strict.40–43 NAATs are the sample of choice for testing asymptomatic patients.40 These techniques show similar sensitivity in urine and urethral specimens from men,43 and also similar sensitivity in endocervical specimens taken from physicians and those self-taken form the patients.44 However, in women, urine samples have lower sensitivity than genital swabs for testing.45 Moreover, NAATs are significantly more sensitive than culture for the detection of NG in pharyngeal and rectal samples,46,47 being the tests of choice for screening for these kind of infections. However, these techniques are not approved for testing specimens from these locations. A summary of the commercially available and FDA (Food and Drug Administration)-cleared NAAT assay platforms for the detection of NG in the United States can be found in the current American guide for the diagnosis of these infections.32

Examination of CSF in a patient with neurologic signs and symptoms must include total protein, number of mononuclear cells and serologic test. CSF laboratory abnormalities (pleocytosis and an increased protein concentration) are common in persons with neurosyphilis. CSF-VDRL is highly specific but insensitive (a positive CSF VDRL test is observed in only about 1:3 cases of neurosyphilis). Rapid plasma reagin testing on CSF is not recommended.48

In a person with neurologic signs or symptoms, a reactive CSF-VDRL (in the absence of blood contamination) is considered diagnostic of neurosyphilis. When CSF-VDRL is negative despite the presence of clinical signs of neurosyphilis, and abnormal CSF cell count and/or protein, neurosyphilis should be considered.

The CSF FTA-ABS test is less specific for neurosyphilis than the CSF-VDRL but is highly sensitive. In patients with suspicion of neurosyphilis but a negative CSF VDRL, a CSF FTA-ABS test can be used to rule out neurosyphilis.48

Because maternal nontreponemal and treponemal IgG antibodies can be transferred from mother to child, treponemal testing of infant serum is not recommended. A fourfold increase or more of the titre of a NTTs in the child’s as opposed to the mother’s serum (both obtained simultaneously at birth, is highly suggestive of congenital syphilis but its absence does not exclude a diagnosis.52 Children from mother with a positive treponemal test for syphilis and any evidence of congenital syphilis on physical examination, or long bone x-ray suggestive, or reactive CSF VDRL assay, or elevated cerebrospinal fluid cell count or protein (without other cause) can support a diagnosis of congenital syphilis.52

Diagnosis of trichomoniasis is commonly based on the microscopy examination of wet preparations of vaginal and urethral discharges, prostatic secretions, and urine sediments. Specimens should be mixed with a drop of physiologic saline (but never refrigerated) and examined microscopically within 1 h under low power (magnification x100), with reduced illumination. The presence of actively motile trichomonas is diagnostic of the infection.60 Polymorphonuclear cells are often present in these preparations. However, although this technique is rapid and inexpensive the sensitivity is between 50-70% and may be less in asymptomatic women. The most important factor affecting the sensitivity of wet mount testing is the time between collection and examination of the specimen.

Culture of genital fluids has been considered the gold standard for diagnosis, although it requires 18-24 h of incubation. The sensitivity of the culture is greater than 80% compared with the wet mount. Amies gel agar transport medium might maintain the viability for culture of TV on swabs held at room temperature for 24 ± 6 h before inoculation of specimen into a culture pouch. However, the best practice is that specimens be collected properly and inoculated immediately into the appropriate medium, such as modified Diamond's, Trichosel, or Holander's medium. Culture systems or systems that allow direct inoculation, transport, culture, and microscopic examination are commercially available.61

Several antigen detection methods have been developed, being the main advantage that they are rapid and easy to perform. A latex agglutination test have demonstrated an excellent sensitivity.62 An immunochromatographic capillary flow assay is commercially available for qualitative detection of TV antigens from vaginal swabs.63,64 The OSOM Trichomonas Rapid Test Kit is a dipstick assay providing results in 10 min. This test has demonstrated good sensitivity and specificity compared with other diagnostic methods.65 A rapid test has been developed for TV detection. This assay uses novel electrochemical end-point detection using a multicopy region of the TV genome as the assay target. The sensitivity and specificity achieved using this assay is comparable with that achieved for existing nucleic acid amplification test (NAATs).66

The Affirm VPIII is a nucleic acid hybridisation test that uses synthetic nucleic acid capture probes and colour development detection probes; compared with NAATs, this technique had a sensitivity of 46%.67

The AmpliVue assay uses isothermal helicase-dependent amplification (HDA) and targets a conserved repeat DNA sequence of TV.68 This technique has been recently approved by the FDA for vaginal swabs. On the other hand, Solana Trichomonas assay is an in vitro qualitative NAAT for the detection of TV also using the HDA technology and the Solana instrument.69 It was recently FD approved. Compared with a NAAT assay, the sensitivity/specificity was 89.7%/99.0% for swabs and 100%/98.9% for urines.

Finally, the GeneXpert TV assay has been approved by the Food Drug Administration (FDA) for use with male urine.

These techniques have now become commercially available for the diagnosis of TV in women and have replaced culture as the gold standard test due to their excellent sensitivity and specificity. Genital and urine samples are acceptable specimens.70 NAATs have not been approved for men by the FDA, but these techniques have shown high sensitivity and specificity for this population. Among women, NAATs may detect a prevalence of threefold to fivefold higher than wet mount microscopy.71

Currently, there are two robotic FDA-approved NAAT platforms for the detection of TV in women: these include the Aptima TV assay (Hologic Gen-Probe)70 and the BD ProbeTec Qx assay on the BD Viper system (Becton Dickinson).72

Studies using the Trichomonas Aptima Combo 2 assay have shown a superior performance compared to other methods. This assay is approved for the detection of TV infections from a wide variety of specimens such as vaginal or endocervical samples, ThinPrep liquid-based cytology samples, and urine specimens.

On the other hand, the BD TV Qx uses female vaginal or endocervical swabs as well as urine and liquid-based cytology. This technique has demonstrated an excellent sensitivity and specificity, and the time of detection is less than 5 h.

Another assay available outside the US is the Seeplex STD 6 ACE detection system (Seegene).73 This assay is a multiplex PCR targeting unique genes of the specific pathogen.

Finally, the BD MAXTM System provides an assay suitable for use with female urine and vaginal or endocervical swab samples. Male urine has not yet been evaluated for TV. This assay has a sensitivity ≥ 91.5% and specificity ≥ 98.6%.74

In the market, there is currently more than 125 techniques for HPV detection. These techniques can be differentiated in four types:

• -

  DNA detection techniques: the HPV DNA is detected from the capside region as well as from the E6 oncogen.

• -

  RNA detection techniques: the RNAm is detected from the HPV E6/7 oncogenes.

• -

  in situ hibridization tecniques: they have low sensitivity and specificity.

• -

  Serological techniques: only used for epidemiological purposes and vaccinal efficacy.

According the technology used, the main commercially available systems may be classified as follows:

• 1

  Signal amplification methods: capture of RNA-DNA hybrids and Chemical Invader.

• 2

  Target DNA amplification methods: PCR, real-time PCR, multiplex PCR, transcription mediated amplication (TMA), dual priming oligonucleotide (DPO) and Matrix-assited laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

In 2009, an international expert committee proposed that any technology must be as accurate as the techniques used at this moment as the gold standard (GP5+/GP6 + PCR and hybrid capture)85 in order to be used in the primary screening of cervix cancer in women. This committee introduced some criteria based on both clinical sensitivity and specificity more than 0,90 and 0,98, respectively.

On the other hand, the VALGENT protocol is an international network for the validation of genotyping HPV assays. Moreover, the FDA approval is achieved when a method establish their sensitivity and specificity by prospective studies performed in three or more sites.

Abbott Real Time HR-HPV and BD Onclarity HPV are two DNA amplification techniques by RT-PCR fully automated. Abbott technology allows the process of the primary tube and reports genotypes 16/18 and others non 16/18 differently. This method is mainly indicated for laboratories with high workload. BD Onclarity use SDA (Strand Displacement Amplification) technology and amplify the E6/7 region.

Anyplex II HPV HR (Seegene) is based on multiplex RT-PCR with DPO and TOCE technology. This method allows the genotyping of 14 genotypes in the same reaction and their relative quantification.

The Xpert HPV genotyping system (Cepheid) is the fastest test (1 h). With this method, we may obtain the genotyping of HPV-16 and other 5 group of high-risk genotypes. Due to their low rate of contamination, it is recommended for laboratories low workload.

Other techniques clinically validated are the virus detection by MALDI-TOF MS and by reverse-hybridization with arrays in microspheres (Luminex).

Linear Array HPV Genotyping Kit (Roche Diagnostics) detects 37 genotypes, very useful for studies of vaccine impact or for epidemiological purposes.

Four techniques are FDA-approved for cytological screening of ASCUS or for screening with cytology and HPV at the same time: hybrid capture (Qiagen), Cervista (Hologic), Cobas 4800 HPV (Roche Diagnostics) and APTIMA (Hologic). Only the Cobas HPV test is approved by FDA for population screening based on HPV detection.

It was the first method approved by the FDA (March 2003) for the detection of HPV oncogenic genotypes. It is a liquid/solid phase signal amplification method based on hybridization in solution of long synthetic RNA probes complementary to the genomic sequence of 13 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and 5 low-risk (6, 11, 42, 43, and 44) HPV types. The hybrids are detected by some reactions that generate a luminescent signal that can be detected by chemiluminiscence. The main limitation of this assay is that does not discriminate the genotype and the cross-reactivity that may lead to false-positive results.86

It was approved by the FDA in 2009. This assay is based on Invader technology consisting of concurrent two-part isothermal reactions. The main reaction detects the presence of specific viral DNA sequences, while the second one generates fluorescence. The presence of any of the 14 high-risk HPV genotypes could be detected (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68), but not in an individualized manner. However, Cervista HPV 16/18 identify HPV 16 and 18 individually.

The Cobas 4800 system is an automated method that uses the primary sample obtained for the liquid-based cytology. The results appear differentiated in four channels: HPV 16, HPV 18, high-risk HPV non 16/18 (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and β-globin (internal control). The main advantages are the high sensitivity, reproducibility, and high-degree of automation.

It is an assay that identify the presence of 14 high-risk genotypes by the identification of the viral RNAm from the E6/7 oncogenes. The presence of transcripts of the HPV oncogenes is a more accurate and specific marker of cell infection or transformation by hihg-risk HPV. This method is useful in differentiating between episomal and integrated HPV oncogenic transcripts, such as in cervical cancer. This technique consist of three steps: capture, amplification by TMA system and detection by hybridization.87 However, the main problem with this technique is that RNA is much more labile than DNA and less available in most biological specimens.

Recent developments in combining molecular probes with silicon-based chips may lead to quick and relatively inexpensive diagnostics. This technology requires the use of silicon chips; the surface of the chip is covered with a fine layer of gold, and molecular probes are attached to the surface of the chip. Each of the molecular probes differs in the DNA target they are designed to hybridize. If binding is detected, the sample would be considered postive for HPV.

The majority of studies employed enzyme immunoassays, but the main problem with the use of the serology is standardization and the establishment of an international standard assigning one unit measure or international unit. Studies have demonstrated that about half of people exposed to HPV never developed measurable titers of antibodies.88

The over-expression of this protein has been proposed as a tissue marker for high-risk HPV infection. Positivity of this protein increases with lesion severity, and a high percentage of HSIL cytology specimens are positive. The sensitivity of P16INK4a assays to detect CIN3 is similar to HPV DNA assays, but the specificity needs to be improved. Its role as a molecular prognosis factor is still an issue pending assessment.89