Real-time PCR as a Diagnostic Tool for Bacterial Diseases

Max Maurin

Disclosures

Expert Rev Mol Diagn. 2012;12(7):731-754. 

In This Article

Diagnostic Applications for Bacterial Diseases

Panbacterial qPCR

Amplification and sequencing of the gene encoding 16S ribosomal RNA using universal primers (panbacterial PCR) has become widely used in clinical laboratories for identification of bacteria at the genus and species levels, either after their isolation in culture or directly from clinical samples.[5] Panbacterial qPCR tests for direct detection of bacterial DNA from clinical samples have also been developed.[5–7] This method allows rapid and wide-spectrum detection of bacteria, especially in patients with negative cultures because of previous administration of antibiotics. It is often successful, provided that a single bacterial species is present in the clinical sample and the bacterial load is high enough to be detected. When multiple bacterial species are present in a clinical sample, a mix of 16S ribosomal DNA (rDNA) sequences will be obtained, and identification of individual species usually necessitates specific techniques (DNA cloning, high-throughput sequencing, ESI mass spectrometry, and so on) that are not yet available in most clinical laboratories. It is to be noted, however, that commercial software potentially allowing specific identification of mixed 16S rDNA sequences is currently sold.[8] Almost all clinical samples can be tested by this method, but those contaminated with a rich and varied commensal flora are not suitable. Examples of possible applications include detection of bacteria responsible for meningitis, endocarditis, arthritis, osteitis and deep abscesses and suppurations.[5–7,9,10] Panbacterial qPCR has also been used for quantification of the bacterial flora, for example, in blood,[11] intraocular samples[12] or chronic wounds.[13]

qPCR for Easy-to-Grow Bacteria

Staphylococcus Aureus. S. aureus is responsible for a wide range of infections in humans including skin and soft tissue infections, arthritis, osteitis, deep abscesses, pneumonia, endocarditis, urinary tract infections, enteritis and bacteremia.[14,15] In addition, methicillin-resistant S. aureus (MRSA) is a major nosocomial pathogen.[14,15] In healthy carriers, S. aureus is found primarily in the nasal cavity. Culture remains the reference method for the screening and clinical diagnosis of S. aureus/MRSA carriage and infection.[15] qPCR tests may be useful for rapid detection of MRSA nasal carriage.[16–21] The cost–effectiveness of this strategy has been debated, and a targeted rather than universal screening is warranted in hospitals with an MRSA incidence lower than 5% of admitted patients.[22] qPCR tests may also be useful for diagnostic purposes.[14] In-house and commercial qPCR tests have been developed for rapid detection of S. aureus/MRSA in clinical samples and blood cultures.[21,23,24]

Streptococcus Pneumoniae. S. pneumoniae is primarily responsible for respiratory tract infections (e.g., pneumonia), meningitis and bacteremia. The natural reservoir of this bacterium is the oropharynx of humans. Therefore, detection of S. pneumoniae using culture or qPCR from respiratory samples may correspond to infection or contamination by the commensal flora. Quantitative sputum cultures are used to tentatively differentiate infection from contamination, and authors have tried to do so using qPCR.[25]S. pneumoniae DNA can also be detected by qPCR in blood samples or in positive blood cultures in patients with bacteremia.[26–30] qPCR is currently used on a routine basis mainly for rapid detection of S. pneumoniae DNA in cerebrospinal fluid in patients with meningitis,[31] and in sputum samples in patients with pneumonia.[26,28] Among gene targets, the lytA and Spn9802 genes have been shown to allow more specific detection of S. pneumoniae than the ply gene.[29,30] qPCR tests targeting the cpsA gene (capsule protein) also allow determination of S. pneumoniae serotype.[26]

Streptococcus Agalactiae. S. agalactiae is responsible for skin and soft tissue infections, urinary tract infections, bacteremia and occasionally severe neonatal infections.[32] Culture remains the reference diagnostic method for most S. agalactiae infections. The prevention of neonatal infections is currently based on the systematic screening of pregnant women for S. agalactiae vaginal carriage (usually by culturing a vaginal swab collected during the eighth month of pregnancy) and antibiotic treatment in those with a positive screening test at the time of delivery.[32] However, approximately 15–20% of parturient women have unknown S. agalactiae carriage status. A number of qPCR tests have been evaluated for rapid per partum detection of S. agalactiae in vaginal and rectal samples of the parturient woman.[33–37] These tests are well-adapted in this situation, allowing rapid and sensitive detection of S. agalactiae, and more appropriate decisions concerning administration of antibiotic prophylaxis.

Streptococcus Pyogenes. S. pyogenes is the primary bacterial cause of pharyngitis.[38] Culturing S. pyogenes from throat samples remains the reference method, allowing evaluation of antibiotic resistances, especially in patients allergic to b-lactams or in case of treatment failure. Many tests allowing rapid detection of S. pyogenes antigens in throat samples are now available.[38] These tests display high sensitivities and specificities (>95%) compared with culture,[38] and although more expensive, they are more suitable for a quick decision regarding the need for antibiotic therapy.[38] Most guidelines still recommend obtaining a throat culture in patients with severe pharyngitis and a negative antigen test because of higher sensitivity of the former technique.[38] qPCR tests for rapid detection of S. pyogenes DNA in throat samples have been developed.[39,40] Their sensitivity is higher than that of culture. However, these tests remain costly and they cannot be used as point-of-care tests, nor for evaluation of antibiotic resistances.

Listeria Monocytogenes. L. monocytogenes primarily causes bacteremia, meningoencephalitis, enteritis and maternofetal infections. This bacterium can be grown from various clinical samples, including blood, cerebrospinal fluid, placenta and fetal or neonatal specimens. A qPCR test was recently developed for rapid detection of L. monocytogenes DNA in the cerebrospinal fluid of patients with meningoencephalitis.[41]

Neisseria Meningitidis. N. meningitidis is a commensal of the oropharynx in humans and is primarily responsible for bacteremia, meningitis and purpura fulminans.[42] Detection of N. meningitidis oropharyngeal carriage is not performed on a routine basis, whereas diagnosis of bacteremia, meningitis and purpura fulminans is usually based on isolation of bacteria from blood, cerebrospinal fluid or less frequently from other clinical specimens (e.g., skin lesion biopsies). qPCR may be useful for early detection of N. meningitidis DNA in these clinical samples, especially in patients with very severe infections (e.g., purpura fulminans).[43–46] Its sensitivity is greater than that of culture in patients who have received antibiotics before clinical sample collection, a common situation for N. meningitidis infections. False-negative results have been reported, however, due to nucleic acid polymorphism in the ctrA gene.[47,48] Finally, specific qPCR tests also allow determination of the involved serogroup,[44,45] thereby guiding vaccine prophylaxis in healthy persons who have been in contact with the patient. Vaccines are available to prevent infections caused by serogroups A, C, Y and W135 strains, but not yet for serogroup B strains, which are most prevalent in many developed countries.

Haemophilus Influenzae. H. influenzae is primarily involved in upper and lower respiratory tract infections (e.g., otitis, sinusitis, epiglottitis, bronchitis and pneumonia), meningitis and bacteremia.[49] Infections caused by H. influenzae serotype b (Hib) predominate and are the more severe, but the systematic use of specific anti-Hib vaccines in infants has dramatically reduced the incidence of these infections in developed countries. Diagnosis relies on culture of this pathogen from various clinical specimens. However, qPCR tests have been developed for rapid detection of H. influenzae DNA, especially in the cerebrospinal fluid and sputum samples from patients with meningitis or pneumonia, respectively.[50,51] Some qPCR tests also allow determination of the capsular type (a to f) of H. influenzae strains.[52] Wang et al. recently developed a new qPCR test allowing better detection of non-b H. influenzae-related infections, a significant advantage in Hib-vaccinated populations where other capsular types may now be more frequently encountered.[50]

Shiga Toxin-producing Escherichia Coli. In recent years, outbreaks of severe enteritis cases caused by Shiga toxin-producing E. coli (STEC) have been described worldwide, with a variable percentage of patients experiencing severe hemolytic and uremic syndrome.[53,54] The involved E. coli strains may be of different pathotypes (enterohemorragic, enteroaggregative) and genotypes, with probable variable virulence potential. However, their most significant virulence factor is the production of the Shiga like toxins (also named Vero-toxins) encoded by the stx1 and stx2 genes. Because STEC are difficult to isolate from the rich and varied intestinal flora and display only few and inconstant phenotypic traits (e.g., sorbitol-negative 'O157' E. coli strains), qPCR tests targeting stx1 and stx2 in stool samples are currently the most efficient diagnostic techniques, allowing a rapid, sensitive and specific diagnosis of STEC-related infections.[55–60] The sensitivity of qPCR tests may be further increased by testing stool specimens that have been previously enriched by short-time culture.[56]

qPCR for Slow-Growing, Fastidious-Growing & Uncultivable Bacteria

Mycobacterium Species. Most mycobacterial species pathogenic for humans are slow-growing microorganisms.[61,62] Also, the traditional methods for their phenotypic identification and antibiotic susceptibility testing are fastidious and time-consuming. The modern diagnostic strategy of mycobacterial diseases includes rapid culture in liquid media (e.g., Middlebrook) using automated systems, direct detection in clinical samples using nucleic acid amplification methods, molecular identification of Mycobacterium spp. from positive cultures or directly from clinical samples, and direct detection of antibiotic resistance genes.[61] qPCR methods have been developed to detect either nontuberculous mycobacteria (NTM)[63–66] or tuberculous mycobacteria.[67–71] Because Mycobacteriumleprae (the agent of leprosy) is an uncultivable microorganism, reverse transcriptase qPCR tests have been developed for simultaneous detection and determination of the viability of this bacterium.[72] qPCR tests for diagnosis of tuberculosis display sensitivities close to that of culture methods for respiratory samples,[67,68,70,71] but their usefulness has also been demonstrated for extrapulmonary specimens (e.g., cerebrospinal fluid, pleural fluid, synovial fluid, lymph node biopsies).[69,70] These tests are more sensitive for smear-positive than for smear-negative samples.[69,71] In-house qPCR tests targeting multicopy DNA such as the IS6110 showed increased sensitivity.[69,73,74] Commercial qPCR tests may not allow identification of all Mycobacterium species, especially for NTM. A number of in-house qPCR tests have been developed for species identification of isolated strains, but few have been evaluated directly on clinical samples.[66]

Chlamydia Trachomatis & Neisseria Gonorrhoeae. C. trachomatis[75] and N. gonorrhoeae[76] are two main etiological agents of sexually transmitted infections (STIs). Both organisms may cause obstetrical complications and infection in the neonate. Culture of N. gonorrhoeae is usually obtained using enriched selective media and allows testing for acquired resistances to antibiotics, especially to β-lactams, tetracyclines and fluoroquinolones.[76]C. trachomatis isolation necessitates eukaryotic cell systems because of its strict intracellular lifestyle.[75] Serological techniques for C. trachomatis infections lack specificity, and these techniques are not useful for N. gonorrhoeae.[75,76] Patients suffering STIs are often coinfected with both pathogens. PCR-based techniques are now considered reference diagnostic methods for C. trachomatis infections, and a number of commercial qPCR tests now allow rapid, sensitive and specific detection of both C. trachomatis and N. gonorrhoeae in urogenital samples.[77–81] qPCR tests should be able to detect the new variant of C. trachomatis with a deletion in the cryptic plasmid.[82]

Chlamydophila (Chlamydia) Pneumoniae & Chlamydophila (Chlamydia) Psittaci. These two species are strict intracellular bacteria responsible for pneumonia.[83,84]C. pneumoniae is responsible for approximately 10% of community-acquired pneumonia, and 5% of bronchitis and sinusitis worldwide, in both adults and children.[83,85]C. psittaci is the agent of psittacosis, a zoonosis occurring in specific endemic areas, especially in persons in contact with infected poultry and in psittacine owners.[84] Culturing of these pathogens in cell systems is tedious, poorly sensitive and even a risk for the laboratory personnel (C. psittaci).[83,84] Serological techniques based on detection of anti-lipopolysaccharide antibodies are not species-specific and only allow a retrospective diagnosis.[83,84] Because C. pneumoniae infections are common, serum residual antibody titers are found in many patients. Compared with serological methods, PCR-based techniques are useful for early diagnosis of Chlamydophila-related infections.[85] Target sequences include a portion of the 16S rDNA,[86,87] the 16S–23S interspacer sequence,[87] outer membrane proteins OmpA[88,89] and OmpB,[90] or the incA and envB genes, respectively encoding inclusion membrane protein A[91] or a cysteine-rich protein.[92] qPCR tests display sensitivities of 10 inclusion forming units (IFU)[89,92] to 0.1–1 IFU.[86,93] A number of commercial or in-house qPCR tests allow rapid detection of C. pneumoniae in respiratory samples from pneumonia patients.[85,86,93–95] However, these tests cannot differentiate chronic oropharyngeal carriage and acute respiratory infection.[95] qPCR tests have also been used for detection of C. pneumoniae in cerebrospinal fluid and vascular tissues in order to tentatively correlate chronic infection with this pathogen and neurological diseases (e.g., multiple sclerosis) or cardiac diseases (e.g., coronary artery disease[88,90]). Home-made qPCR tests have been developed for specific detection of C. psittaci in respiratory samples.[87,89,91,92]

Rickettsia. Diagnosis of spotted fever group rickettsiosis (e.g., Rocky Mountain spotted fever caused by Rickettsia rickettsii, and Mediterranean spotted fever caused by Rickettsia conorii), typhus (epidemic typhus caused by Rickettsia prowazekii and murine typhus caused by Rickettsia typhi) and scrub typhus (Orientia tsutsugamushi) relies on serological methods.[96–98] These tests primarily detect anti-lipopolysaccharide antibodies and cannot accurately determine the involved Rickettsia species. This may be obtained using western blot and adsorption methods. Culturing of Rickettsia spp. in cell systems is poorly sensitive and only performed in reference laboratories. qPCR methods have been developed for detection of rickettsial DNA from skin biopsies and blood or serum samples,[96–99] allowing early confirmation of diagnosis, whereas serological tests are positive only 2–3 weeks after symptom onset. Identification of the rickettsial species involved may be obtained using specific primers and probes[97,99] or by sequencing amplified DNA fragments.[96,97,99]

Coxiella. Coxiella burnetii, a strict intracellular bacterium, is responsible for the zoonosis Q fever.[100] Most patients present with acute manifestations, mainly a influenza-like illness, pneumonia or hepatitis, or occasionally severe forms such as myocarditis and encephalitis. A few patients suffer from chronic diseases, including endocarditis, chronic vascular infections and maternofetal infections. Serological tests allow accurate diagnosis of Q fever and differentiation of acute from chronic infections. Culturing of C. burnetii in cell systems is poorly sensitive and only performed in reference laboratories. qPCR tests allow detection of C. burnetii DNA from blood or serum samples in patients with acute Q fever at an early stage of the disease, before serological tests become positive.[101,102] qPCR tests are also useful in patients with chronic Q fever, for rapid and sensitive detection of C. burnetii DNA in blood and serum samples, cardiac vegetations, vascular tissues (e.g., aortic aneurysm) and thrombus, and placental tissues in case of maternofetal infection.[100] Surprisingly, false-positive results were recently reported by Tilburg et al. when using a commercial PCR Master Mix containing C. burnetii DNA, probably as a result of contaminated bovine serum albumin.[103]

Ehrlichia & Anaplasma. Anaplasma phagocytophilum and Ehrlichia chafeensis are respectively the etiological agents of granulocytic and monocytic ehrlichiosis in humans.[104] These Gram-negative, strictly intracellular bacteria have a wide animal reservoir. Humans are contaminated through tick bites. Both diseases may manifest by influenza-like symptoms, including fever, myialgia, arthralgia, headaches and a rash. Severe forms may occur, especially in patients involved with granulocytic ehrlichiois because of severe granulopenia. Diagnosis primarily relies on serological techniques. A. phagocytophilum morulae are occasionally visualized in polymorphonuclears on blood smears. Real-time PCR tests have been developed for direct detection of these bacteria in blood samples.[105–107] Although both sensitive and specific, the use of these tests is currently restricted to reference laboratories.

Bartonella. Bartonella species are Gram-negative bacteria with a large and diverse animal reservoir, except Bartonella quintana, which is restricted to humans.[108]B. quintana and Bartonella henselae (a cat species) are the two main human pathogens worldwide.[108,109]B. quintana is transmitted from human to human via the body louse. Human infections with B. henselae occur after cat bites or scratches, or via the bite of cat arthropods. Both species are responsible for bacteremia, endocarditis and prolonged fever in humans. Bacillary angiomatosis may occur in immunocompromized patients. B. henselae is also responsible for cat-scratch disease, a chronic regional lymphadenopathy occurring after skin inoculation of the bacterium. Bartonella are fastidious and slow-growing bacteria, and thus culture cannot be considered a reference diagnostic technique because of its low sensitivity.[108,109] Serology is useful in immunocompetent patients, whereas it is usually negative in immunocompromized ones.[108,109] Real-time PCR methods have been developed to detect Bartonella DNA in clinical samples, especially in lymph node specimens, in cardiac vegetations and in angiomatosis lesions.[106,110–114] Although B. henselae and B. quintana are most often involved in human infections, other Bartonella species may cause similar clinical manifestations, especially chronic lymphadenopathies.[108] Thus, developed qPCR tests usually target most Bartonella species.[106,110–114] Target DNA sequences include NADH dehydrogenase gene,[110]groEL,[111]pap31,[112] citrate synthase gene (gltA)[113] and the riboflavine synthase gene.[114] All these tests are considered highly specific for Bartonella spp. Their sensitivities are difficult to evaluate because of current lack of a reference diagnostic test.[106,110–114]

Mycoplasma Pneumoniae. M. pneumoniae is primarily responsible for community-acquired pneumonia, acute pharyngitis, tracheobronchitis and bronchiolitis.[115]M. pneumoniae is a fastidious and slow-growing bacterium, and thus culture lacks sensitivity and is not well-adapted for rapid diagnosis of infections caused by this microorganism. Serological tests are often used to confirm M. pneumoniae infection. In particular, ELISAs are available for detection of IgM or IgM/IgG antibodies.[115] The IgM antibodies may be detected as early as the seventh day following symptom onset, but IgM detection both lacks sensitivity and specificity.[115] A fourfold increase in antibody titers is more specific, but it is usually obtained only after 2–3 weeks evolution of the disease.[115] qPCR detection of M. pneumoniae DNA in respiratory samples allows early diagnosis.[116–121] These tests are less sensitive but more specific than serological tests,[119] although some positive tests may only correspond to asymptomatic carriage of M. pneumoniae in the upper respiratory tract. Thus, M. pneumoniae infections may currently be best diagnosed using a combination of serological and qPCR tests.[119] Using P1 gene typing, qPCR tests may allow differentiation of the two genotypes of M. pneumoniae,[122] and even variants of each of these two genotypes.[123]

Urogenital Mycoplasma & Ureaplasma Species.Mycoplasmahominis, Mycoplasmagenitalium, Ureaplasmaurealyticum and Ureaplasmaparvum are responsible for urogenital infections in men (nongonococcal urethritis, epididymoorchitis, acute pyelonephritis) and women (urethritis, vaginosis, cervicitis, salpingitis, pelvic inflammatory disease, endometritis, chorioamniotitis and maternofetal infections), although the pathogenicity potential greatly varies between species.[124–128]Mycoplasma and Ureaplasma may also cause wound infections, arthritis, pneumonia, mediastinitis, pericarditis, endocarditis, osteitis, arthritis, peritonitis and bacteremia, especially in immunocompromized persons.[125,128] The pathogenic role of other species such as Mycoplasma fermentans, Mycoplasma penetrans and Melongena pirum has been recently discussed. Culture is considered the reference method for diagnosis of M. hominis, U. urealyticum and U. parvum infections, although the two later species cannot be differentiated on phenotypic traits. This technique is not adapted for diagnosis of M. genitalium infections because of the fastidious nature of this bacterium.[129] qPCR techniques are well adapted for diagnosis of genital and extragenital infections caused by Mycoplasma[124–126,129,130] and Ureaplasma species.[127,128,131] They allow rapid detection of these bacteria in clinical samples, and their differentiation at the species level (including U. urealyticum and U. parvum). As for culture, a major limitation of these tests is that the detection of Mycoplasma and Ureaplasma on the genital mucosa in women (especially in vaginal and cervical swabs), and more rarely in men, may represent only healthy carriage. Only qPCR tests allow diagnosis of M. genitalium infections at present.[129,130] These tests have helped better define the pathogenic role of this fastidious species.[132]

Helicobacter Pylori. H. pylori is the main etiological agent of idiopathic peptic ulcer and it occasionally causes malignant diseases such gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma.[133] There are a number of invasive (necessitating upper endoscopy) or noninvasive techniques allowing diagnosis of H. pylori infection.[133] Among noninvasive techniques, the breath test and direct detection of H. pylori in stools are the most useful, the first one allowing evaluation of antibiotic treatment efficacy. Culturing H. pylori from gastric or duodenal biopsies is needed in case of treatment failure to check for antibiotic resistances in the involved strain. However, H. pylori culture is tedious, time-consuming and lacks sensitivity. PCR-based techniques, especially qPCR, are useful to detect H. pylori in gastroduodenal biopsies and for simultaneous detection of 23S rRNA gene mutations responsible for macrolide resistance in this species.[133–135]H. pylori DNA may also be detected in stool specimens.[136]

Clostridium Difficile. C. difficile is commonly found in the intestinal bacterial flora of humans, but only rarely causes C. difficile-associated diarrhea and pseudomembranous colitis, usually in patients receiving a broad-spectrum antibiotic therapy.[137] Both diseases are caused by C. difficile toxins A and B, respectively encoded by the tcdA and tcdB genes, whose expression is under the control of the tcdC repressor gene. Virulent C. difficile strains harboring the tcdB, but not the tcdA genes have been described. The microbiological diagnosis of C. difficile infection primarily relies on direct detection of toxins A and B in patients' feces, using the reference cell culture cytotoxicity neutralization assay or ELISA tests.[137] Because the cell culture cytotoxicity neutralization assay is fastidious and ELISA tests lack sensitivity, these tests are usually combined with direct detection of C. difficile in stool samples, including C. difficile glutamate dehydrogenase detection (GDH test) and toxigenic cultures.[137] Both tests lack specificity because detecting C. difficile may only correspond to bacterial carriage. Also, the GDH test does not differentiate toxin-producing from toxin-nonproducing (nonpathogenic) C. difficile strains, and the toxigenic culture method is tedious and time-consuming. qPCR tests are designed to rapidly and specifically detect toxigenic C. difficile strains in stool samples. They should detect the chromosomal gene tcdB, whereas some also detect tcdA, the binary toxin gene (cdt) and the tcdC gene deletion at nucleotide 117 found in the highly virulent 027/NAP1/BI strains.[138–143] A current recommended diagnostic algorithm consists of screening stool samples using a sensitive antigen assay (e.g., GDH test) and confirming positive samples by a toxin PCR/qPCR test.[144] This strategy is well suited for a quick decision on whether or not to isolate the patients.

Legionella. Legionella pneumophila is responsible for approximately 90–95% of legionellosis cases, and its serogroup 1 for approximately 90% of those cases.[145] Legionellosis may also be caused by other L. pneumophila serogroups and other Legionella species (e.g., Legionella micdadei, Legionella longbeachae, and so on). Legionella spp. are fastidious slow-growing bacteria. Only approximately 15–20% of legionellosis cases are diagnosed by culture, but this may reflect the lack of attempt to culture this pathogen rather than difficulties in its isolation.[145] Serology is rarely useful because many patients harbor specific serum residual antibody titers, and a significant antibody response is only mounted after 3–4 weeks evolution of the disease. Thus, most cases of legionellosis are currently diagnosed by detection of L. pneumophila antigen in patients' urine – that is, the urinary antigen test (UAT).[145] These tests primarily target L. pneumophila serogroup 1 antigen, but can occasionally detect other serogroups of this species.[145] qPCR tests have been evaluated for direct detection of Legionella DNA in respiratory samples, or less frequently in blood and urine samples.[146–149] These qPCR tests usually allow detection of all L. pneumophila serogroups or even all Legionella species.[147–149] More recently, a qPCR test allowing specific detection of serogroup 1 strains of L. pneumophila was developed.[146] Legionellosis diagnosis is currently most often determined by the UAT than by qPCR tests in respiratory samples.[145] The main limitations of the later tests are the possibility of false-negative results due to the presence of DNA polymerase inhibitors in sputum samples,[150] and difficulties in obtaining appropriate lower respiratory tract samples, especially in women and young children. Occasionally, however, positive qPCR tests have been found in patients with a negative UAT.[147] Thus, combining the UAT and qPCR tests could be useful to increase the overall sensitivity of legionellosis diagnosis.[149]

Bordetella. Bordetella pertussis and Bordetella parapertussis are etiological agents of pertussis (also referred to as whooping cough).[151] The disease remains prevalent in countries where pertussis vaccination is not implemented, but also in countries where the population is vaccinated because available vaccines provide neither a complete nor a lifelong protection against the disease. Both bacterial species can be cultured from respiratory samples, but this technique lacks sensitivity, especially when respiratory samples are collected more than 2–3 weeks after symptom onset.[151] Serological techniques have long been used to diagnose whooping cough, but they only allow a late diagnosis.[151] qPCR tests have now replaced traditional diagnostic techniques because of their higher sensitivity and specificity, and the possibility for early diagnosis.[152–157] These tests should allow accurate differentiation between B. pertussis and B. parapertussis-related infections, the former being usually more severe. This was not obtained when using some target genes such as the pertactin gene.[158] The IS481 and IS1001 gene sequences are often used for detection of B. pertussis and B. parapertussis, respectively, because a large number of copies of these insertion sequences are present in the Bordetella genomes.[152–157] However, false positive results have been observed with the IS481 target for Bordetella holmesii and Bordetella bronchiseptica strains[151] and IS1001 target for B. bronchiseptica.[152] The ptxS1 gene encodes pertussis toxin subunit 1 and thus is not a species-specific target.[152] The ptxA-pr encoding B. pertussis toxin subunit 1 promoter[155] and two DNA regions referred to as BP283 and BP485[156] were found to be more specific targets for this species. As for culture, the sensitivity of qPCR decreases over the duration of the illness, but its sensitivity is less affected by antibiotic treatment.[151] Also, a lower bacterial load was found in adults compared with children, suggesting lower sensitivity of qPCR tests in the former group of patients.[159]

Corynebacterium Diphtheriae. C. diphtheriae and less frequently Corynebacterium ulcerans are etiological agents of diphtheria, a toxin-mediated disease.[160] The disease has been controlled in most developed countries due to specific vaccination (toxoid) usage, but large outbreaks have occurred in countries where vaccination was abandoned, such as in the states of the former USSR.[161] Diphtheria remains a prevalent disease in many developing countries, where it is still responsible for high mortality rates in children. C. diphtheria and C. ulcerans (a zoonotic pathogen) can be grown from nasopharyngeal specimens in specific media. Only toxin-producing strains cause diphtheria. Detection of antitoxin antibodies in patients' serum serves to evaluate protection against severe forms of the disease, especially in previously vaccinated patients. qPCR tests have been developed for rapid detection of C. diphtheriae and C. ulcerans from clinical samples, especially the tox gene encoding the diphtheria toxin.[160,162,163] Most PCR and qPCR tests can detect both species, although exceptions have been reported.[163] Because C. diphtheria and C. ulcerans tox genes differ from each other in approximately 5% of their base pairs, specific qPCR tests have been developed to differentiate both targets.[160,162] Early diagnostic confirmation of diphtheria is essential for rapid administration of the specific antitoxin treatment, which determines patients' prognosis.

Brucella. Brucella melitensis, Brucella abortus and Brucella suis are responsible for the majority of human brucellosis cases in the world.[164] The disease has been eradicated in a few countries due to large vaccination campaigns in livestock, but remains prevalent worldwide. Brucellosis may occur after direct contact with infected animals or via consumption of contaminated dairy products. The disease primarily manifests by a bacteremia of intestinal origin, and then evolves to a subacute phase, with persistent or relapsing bacteremia, and in some patients to a chronic infection. During the subacute and chronic phases of brucellosis, patients often experience secondary septic localizations, including osteoarthritis, spondylitis, hepatic or splenic abscesses, neurological and cardiac involvement. Diagnosis of brucellosis primarily relies on blood cultures, which are positive in 90% or more patients in the acute phase, and still 50% or more cases during the subacute phase.[164] Serological tests are only useful in patients with negative Brucella cultures, but these tests lack sensitivity and specificity.[164] qPCR tests have been developed to detect Brucella DNA in blood and serum samples in bacteremic patients,[165–168] and in other clinical samples (e.g., bone marrow, synovial fluid, bone biopsies, hepatic and splenic abscesses, cardiac vegetations and cerebrospinal fluid[165,167]). They are more sensitive than culture to confirm localized forms of brucellosis.[165,167]

Francisella. Francisella tularensis subsp. tularensis (type A strains) and subsp. holarctica (type B strains) are responsible for tularemia, a zoonosis restricted to the northern hemisphere.[169] The disease most often manifests by a localized lymphadenopathy in the territory of a skin inoculation lesion, for example, following manipulation of an infected animal (especially hares) or related to an arthropod bite (usually a tick). Infections through the conjunctival or oral routes may lead respectively to the oculoglandular form, or oropharyngeal and intestinal forms of tularemia. Inhalation of F. tularensis aerosols is responsible for the pneumonic form. Whatever the mode of contamination, patients may suffer from a severe illness with neurologic manifestations, referred to as the typhoid form. Tularemia diagnosis is usually confirmed by serological tests.[169] Positive cultures are rarely obtained, and usually correspond to positive blood cultures in patients with bacteremia.[169] qPCR tests are useful to establish a rapid diagnosis at an early stage of the disease,[169,170] whereas serological tests are negative, and to confirm diagnosis at a later stage.[169,171]F. tularensis DNA may be detected in various clinical samples, including in blood and serum samples, conjunctival and pharyngeal swabs, skin lesion exudates, sputum samples and lymph nodes. Ideally, these tests should differentiate type A from type B strains,[171] and these subspecies from the aquatic subspecies F. tularensis subsp. novicida,[172] which has only low virulence potential in humans.

Leptospira. Leptospira interrogans is the etiological agent of leptospirosis, a zoonosis of worldwide distribution.[173,174] Humans may be infected through direct contact with infected animals (especially rats) or more frequently after contact with water contaminated with the urine of these animals. The acute phase of the disease corresponds to an influenza-like illness with bacteremia. Severe complications may occur (e.g., hepatitis, renal failure, neurological and cardiac involvement and so on), especially in patients infected with the more virulent serovars. Because culture of Leptospira sp. is tedious, poorly sensitive and risky for the laboratory personnel, diagnosis is usually made using serological techniques (especially ELISA and microagglutination tests[174]). qPCR tests allow detection of Leptospira DNA in clinical samples (especially blood and serum samples) in the early phase of the disease, before antibody titers are at detectable levels.[173–177] These tests amplify target genes present only in pathogenic strains of Leptospira sp.

Treponema Pallidum. T. pallidum is the agent of the sexually transmitted disease syphilis.[178] The disease typically evolves into different phases, including primary syphilis, secondary syphilis, latent phase and tertiary syphilis. T. pallidum is responsible for obstetrical complications (abortion, stillbirth and neonatal death) in the pregnant woman and congenital syphilis in neonates born from infected mothers. Culturing this bacterium in axenic medium is still not possible and dark-field microscopy examination of clinical samples may give both false-negative and false-positive results. Serology, including treponemal and nontreponemal tests, remains the reference diagnostic method for syphilis.[179] However, diagnostic confirmation may be difficult in the early phase of the disease, before a significant serological response has been mounted, and in some patients with nonspecific secondary or tertiary clinical manifestations.[178,180] qPCR tests allow sensitive and rapid detection of T. pallidum DNA in syphilitic ulcers.[178,180] qPCR tests are much less sensitive and their use has not been fully validated for diagnosis of late manifestations of syphilis and latent syphilis.[178,180,181] Occasionally, however, they allow detection of T. pallidum in blood samples and cerebrospinal fluid of infected patients during the secondary and latent phases of the disease, and in infected pregnant women and neonates.[178,181]

Borrelia. Borrelia burgdorferi, Borrelia afzelii and Borrelia garinii are responsible for most cases of Lyme disease.[182] These spirochetes are transmitted to humans via tick bites. The disease should be diagnosed during the acute phase (a influenza-like illness with rapid onset of the typical erythematic chronicum migrans) to prevent evolution to complications, including articular, neurological and cardiac involvement. Culturing of these species is a tedious, lengthy, poorly sensitive and risky procedure for the laboratory personnel. Serological tests are reference tests, including ELISA and western blot techniques.[182] False-positive results may be found with ELISA tests due to antigenic cross reactions (especially with other spirochetes) whereas immunoblots are more specific, especially those using recombinant proteins.[182] High antibody titers may persist for a prolonged period after antibiotic treatment, which may preclude accurate diagnosis of a reinfection.[182] In patients with skin lesions, qPCR testing of a skin biopsy allows early detection of Borrelia DNA, whereas serological tests are usually negative at this stage.[183] The number of Borrelia sp. in other infected tissues and body fluids is generally very low.[182] qPCR tests usually detect Borrelia DNA in synovial fluid or synovial membrane biopsies of affected joints.[182] Tests using the OspA-encoding gene as the target were reported to be more sensitive than those using the 16S rDNA.[182] By contrast, qPCR tests cannot detect Borrelia DNA in the cerebrospinal fluid of most patients with neuroborreliosis.[184] Low sensitivities have also been reported for blood and urine samples.[182] qPCR tests allow easier determination of the Borrelia species involved.[185]

Bacillus Anthracis. B. anthracis is responsible for anthrax, a zoonosis that may manifest by skin lesions (cutaneous anthrax), by oropharyngeal and digestive symptoms in case of infection through the oral route, by a severe pneumonia in case of inhalation of a contaminated aerosol, and by septicemia.[186] The bacterium may be isolated from various clinical samples (e.g., blood, sputum, cutaneous exudates or biopsies), but sensitivity of sputum culture is low.[186] Cultures should be handled in a biosafety level-3 laboratory because of the high risk of laboratory-acquired infection. Serological tests may detect specific antibodies (e.g., antibodies against edema and lethal toxins, IgG against B. anthracis protective antigen) 2–3 weeks after symptom onset.[186] Both diagnostic techniques are not adapted to the urgent situation of inhalational anthrax. qPCR tests have been developed for early diagnosis of anthrax, which is essential to improve prognosis of patients with systemic forms of the disease.[187,188]

Yersinia Pestis. Y. pestis, the agent of plague, is a zoonosis still present in specific geographic areas, including in Asia, Africa and America.[189] Plague may manifest by a regional lymphadenopathy (bubonic plague), by pneumonia (pneumonic plague) or by a fulminant bacteremia (septicemic plague[189]). The bacterium may be isolated from various clinical samples (e.g., blood, sputum, bubo aspirate). Cultures should be handled in a biosafety level-3 laboratory because of the high risk of laboratory-acquired infection. Serological tests may detect specific antibodies against F1 antigen 1–2 weeks after symptom onset. A few qPCR tests have been evaluated in humans for early diagnosis of plague. Most of them target virulence plasmid genes, including the pla gene encoding a plasminogen activator, which allows highly sensitive detection of Y. pestis in clinical samples.[190,191]

Tropheryma Whipplei. T. whipplei, the agent of Whipple disease, may cause chronic clinical manifestations, mainly chronic diarrhea with malabsorption, neurological involvement (encephalitis) and cardiac involvement (endocarditis[192]). Isolation of this pathogen has been recently obtained in cell culture systems, but this technique remains tedious and poorly sensitive. Specific antibodies may be found in patients' serum, but reliable serological tests are not yet available. PCR-based methods, especially qPCR, are thus the mainstay in diagnosis of Whipple disease together with pathological examination of the involved tissues (especially using periodic acid Schiff stain[192]). T. whipplei DNA may be detected in a number of clinical samples, including stool samples, intestinal biopsies, cardiac vegetations and the cerebrospinal fluid of involved patients. Because many patients may be transiently colonized with this bacterium in their gut, positive PCR tests for intestinal samples should be interpreted with caution.[193]

Multiplex qPCR for Diagnosis of Syndromes & Other Specific Clinical Situations

Bacteremia. Bacteremia is an urgent situation that needs rapid detection and identification of the involved bacterial species in order to optimize antibiotic therapy and improve patient's prognosis.[194] A limited number of species cause the majority of bacteremia cases, including E. coli, S. aureus and S. pneumoniae. The LightCycler Septifast test (a multiplex qPCR test developed by Roche Diagnostics, Basel, Switzerland) allows the detection and identification of 20 different pathogens directly in blood samples.[195–198] Data obtained in real clinical situations have been disappointing, with both false-positive and false-negative results compared with blood cultures.[195,198] qPCR tests have been successfully used for rapid identification and evaluation of specific antibiotic resistances in bacteria grown in blood cultures.[27,199] Multiplex PCR diagnosis of bacteremia is a rapidly evolving field, and a molecular diagnostic approach of bacteremia seems already promising at least in specific patients.[196]

Meningitis. Multiplex qPCR tests have been developed for diagnosis of bacterial meningitis.[200–202] The primary targets are S. pneumoniae, N. meningitidis and H. influenzae. These tests may also allow determination of N. meningitidis serogroup for prophylactic vaccination purposes.[200] These tests are particularly useful in patients with negative cultures because of previous antibiotic therapy.[201]

Endophthalmitis. Bacterial endophthalmitis is a rare, but urgent situation necessitating early diagnosis and treatment to prevent loss of vision of the infected eye.[203] Postsurgical endophthalmitis cases are becoming more common, especially because of increasing frequency of surgical interventions for cataract removal. Postsurgical endophthalmitis is most often caused by coagulase-negative staphylococci (especially S. epidermidis) and oropharyngeal Streptococcus spp., whereas S. aureus, S. pneumoniae and Gram-negative bacteria are less frequently encountered.[203] Isolation of bacteria from the aqueous humor or vitreous samples is the main diagnostic method, but culture sensitivity is only approximately 50–60%.[203] The combination of culture and PCR allows increasing diagnostic sensitivity to 70–80%.[203] More recently, qPCR tests have been developed for direct detection of bacterial DNA in intraocular samples.[204–206] A more rapid and sensitive diagnosis of endophthalmitis may improve patients' care, including administration of optimized local and systemic antibiotic therapy and more rapid decision-making for pars plana vitrectomy.

Pneumonia. A number of bacterial species cause community-acquired pneumonia, including S. pneumoniae, H. influenzae and the atypical pathogens C. pneumoniae, M. pneumoniae and L. pneumophila.[207]S. pneumoniae and L. pneumophila are usually involved in the more severe cases necessitating hospital admission and specific diagnostic procedures for optimal antibiotic therapy. A number of in-house and commercial multiplex qPCR tests can detect these bacterial species as well as many viruses.[208–212] A multipathogen detection approach may also be obtained using the TaqMan low-density array technology.[213] The molecular diagnosis of nosocomial pneumonia, especially ventilator-associated pneumonia, is much more problematic since many bacterial species may be involved.[214] No commercial test is currently available, whereas a rapid etiological diagnosis allowing early optimization of the antibiotic therapy is essential to improve patients' prognosis.

Enteritis. Culture of enteropathogens from stool samples in patients with enteritis remains fastidious and poorly susceptible.[215] The clinical symptoms are not specific and patients with severe diseases may necessitate the administration of a pathogen-specific antibiotic therapy. Multiplex PCR and qPCR tests are well-adapted for rapid etiological diagnosis in patients with enteritis, allowing simultaneous detection of multiple pathogens, including Salmonella sp., Shigella sp., Campylobacter sp., Yersinia enterocolitica, STEC and C. difficile.[215,216] These tests are more sensitive than culture.[215,216] In addition, they allow a significant increase in the detection of multiple enteric pathogens in a single patient.[215]

Sexually Transmitted Infections. Most bacterial species responsible for STIs are fastidious in nature and thus difficult to isolate. Multiplex PCR or qPCR tests may allow simultaneous detection of C. trachomatis, N. gonorrhoeae, M. hominis, M. genitalium, U. urealyticum and U. parvum. They are well-adapted for rapid and sensitive diagnosis of STIs, and perform equally well as the monoplex qPCR tests.[217–219] They also allow increased detection of patients infected with multiple STI pathogens.[218,219]

Chronic Lymphadenopathy. Chronic lymphadenopathy is a common cause of medical consultation, and may correspond to various diseases, including infectious diseases, autoimmune disorders, histiocytosis and malignancies.[220] Early etiological diagnosis is warranted in order to quickly detect life-threatening diseases. Among infectious causes, a number of bacterial species may induce regional or generalized lymphadenopathies. Cultures from lymph node aspirates or biopsies may allow isolation of the involved pathogen, whereas serological techniques are more appropriate for fastidious species. PCR-based techniques have been developed for rapid detection of the later species, including those belonging to the Mycobacterium tuberculosis complex, NTM, B. henselae (the agent of cat-scratch disease) and F. tularensis (the agent of tularemia[220,221]).

Biothreat Agents. An epidemic situation that may evoke intentional release of a CDC class-3 pathogen in the general population will necessitate rapid intervention for early characterization of the involved pathogen and implementation of adapted therapeutic and prophylactic measures.[222] Multiplex qPCR-based diagnosis is the most adapted response, allowing detection of the main agents that may be used for bioterrorism purposes: B. anthracis, Y. pestis, F. tularensis, B. melitensis, Rickettsia sp., C. burnetii and so on. Because any of these pathogens may be responsible for unspecific clinical manifestations, multiplex qPCR tests have been developed for their simultaneous detection.[223–225]

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