Pathogenic
Humans are mainly infected by exposure to urine of infected animals or contaminated environments [1, 2]. The clinical manifestations of leptospirosis range from milder anicteric leptospirosis to severe illness called icteric leptospirosis or Weil's disease. In the mild form, patients present with acute undifferentiated fever, that is mild flu-like illness similar to other tropical infectious diseases such as malaria, dengue fever, enteric fever, typhus, influenza, and hepatitis [1, 13, 14]. Severe cases may present with multi-organ failure, such as jaundice, renal failure, lung hemorrhage, and septic shock [7, 15]. Prompt antibiotic treatment can reduce bacterial burden in patients. Inaccurate or delayed diagnosis leads to serious complications or even fatality. Currently, several diagnostic methods including direct examination with dark-field microscopy, culture, serological tests, and genomic DNA detection by molecular methods have been utilized by diverse laboratories based on their resource settings [3, 15]. This review provides updated information of leptospirosis diagnostic assays, especially rapid identification of pathogenic
Anicteric leptospirosis usually presents as a biphasic illness, i.e., the primary septicemic phase during the first week of infection followed by the secondary immune phase [1, 2, 16]. After acquisition through cracked skin or mucous membrane, pathogenic
Previous studies suggested that whole blood and serum are more suitable for molecular detection than buffy coat [20, 21, 22]. Leptospires can be engulfed by leukocytes and enriched in a buffy coat [23]. However, the buffy coat is not generally used in routine diagnostic laboratories because of an additional step for isolation and higher risk of contamination with chemical and biological inhibitors than serum [20, 22, 24, 25]. Serum is less contaminated with inhibitors, e.g., heme, anticoagulants, and host cell DNA than whole blood [20, 26, 27, 28]. Nevertheless, whole blood samples containing both free-living and phagocytosed leptospires might be better than serum because bacteria are in total components of blood in contrast to serum from which bacteria could be trapped in blood clot after separation [21, 29, 30]. High-performance DNA extraction methods with additional step of inhibitor removal for whole blood may improve diagnostic sensitivity and accuracy.
In cases of leptospirosis with acute meningitis or encephalitis syndrome, leptospiral DNA might be detected in the cerebrospinal fluid (CSF) [25]. Some patients had higher bacterial loads in CSF than in blood [25, 32].
Patients with signs and symptoms compatible with leptospirosis should be confirmed by laboratory tests using microbiological and serological techniques [7, 15]. The specimen collection and assay selection are based on symptomatic period and laboratory performance.
A simple and rapid method to determine leptospiral infection is a direct examination of the pathogen in clinical specimens such as blood and urine under dark-field microscopy.
Microscopic agglutination test (MAT) has been widely accepted as a standard method for confirmation of leptospirosis [38]. This method detects specific agglutinating antibodies in patient sera against reference serovars of
The enzyme-linked immunosorbent assay (ELISA) to measure specific IgM may have higher sensitivity than MAT for diagnosis of acute leptospirosis [39, 40, 41, 42, 43, 44, 45, 46, 47]. Whole-cell
The rapid immunoassays have been used as diagnostic tests for leptospirosis. Agglutination and immunochromatography to detect antibodies, mostly IgM, against leptospires are the key principles of most available kits. They are fast, simple, and user-friendly, thus they can be used for point-of-care testing. However, various studies revealed inconsistent sensitivity and specificity [48, 49, 50, 51, 52, 53]. The antibody baseline varies in different endemic areas, thus local validation is required. Recent study evaluated commercially available rapid diagnostic tests to detect IgM against
Detection of leptospiral antigens should be a better rapid detection platform in the acute phase of leptospirosis. Our group is developing a lateral flow assay targeting LipL32 antigen. The sensitivity and specificity are acceptable in a pilot study. The product has been patented and will be validated for the field use.
Currently, the affordable price of equipment and reagents promotes the use of molecular techniques for diagnosis of infectious diseases. Laboratory capacity has been gradually expanded in developing countries. DNA detection technology methods especially PCR-based methods have been applied for rapid detection of many emerging and re-emerging infectious diseases and have the potential to become a point-of-care testing in endemic areas.
In the early leptospiremic phase, serological assays for antibody detection are limited [1, 2, 16]. Although eradication of leptospires by either immune response or antibiotic treatment results in failure of bacterial culture, genetic materials of leptospires may still be maintained at different sites in patients depending on the course of infection [20, 55, 56]. The molecular assays can detect both live and dead leptospires in various clinical specimens from both phases of leptospirosis leading to increased sensitivity of detection. The common targets for leptospiral detection are located on their housekeeping genes [57, 58, 59] and pathogen-specific regions [29, 30, 59, 60, 61, 62]. The amplification and detection systems included conventional PCR, real-time PCR, and real-time reverse transcriptase PCR (real-time RT-PCR) [57, 63, 64, 65, 66]. Molecular diagnosis of human leptospirosis is summarized in
Molecular diagnosis of human leptospirosis
SYBR green | 60 GE/PCR | Whole blood | 67.7 | 90.0 | [63, 71, 72, 73] | |
103 cells/mL | ND | ND | ND | |||
102 cells/mL, 3 GE/PCR | Serum | 30 | 100 | |||
TaqMan | 40 cells/mL, 1 GE/PCR | Whole blood | 43.0–60.6 | 93.0–99.0 | [21, 60, 74, 75] | |
Serum | 29.1 | 99.0 | ||||
Blood culture | 86 | 100.0 | ||||
Urine | ND | 100.0 | ||||
40 cells/mL, 1 GE/PCR | Whole blood | 18.4–96.4 | 90.0–99.5 | [20, 74, 75, 76, 77] | ||
Serum | 50.0–51.0 | 99.2 | ||||
Buffy coat | 35.7 | 99.7 | ||||
Blood culture | 69.5–100.0 | 95.2–97.0 | ||||
Urine | 39.1 | 91.5–92.5 | ||||
ND | Serum | 53.9 | 99.6 | [22] | ||
Buffy coat | 58.8 | 99.9 | ||||
Urine | 45.0 | 99.6 | ||||
RT-PCR | 1 cell/mL | Whole blood | 64.0 | 100.0 | [66] | |
ddPCR | 10 cells/mL, 1 GE/PCR | Spiked whole blood | ND | ND | Our study | |
RPA | ≤2 GE/Rx | Serum | 94.7 | 97.7 | [78] | |
LAMP | 10 GE/Rx | Serum, urine | 91.67 | 100 | [79] | |
10 GE/Rx | Serum | 43.6 | 83.5 |
SYBR green, SYBR green real-time PCR; TaqMan RT-PCR, TaqMan real-time PCR; RT-PCR, reverse transcription PCR; ddPCR, droplet digital PCR; RPA, recombinase polymerase amplification; LAMP, loop-mediated isothermal amplification; ND, not determined.
cells/mL,
Conventional PCR was initially developed for the detection of leptospires in various clinical samples such as blood, CSF, and urine [64, 65, 67, 68, 69]. A previous study reported excellent specificity (100%) and fair sensitivity (62%) of PCR in serum samples from acute phase of leptospirosis [70]. In this study, PCR was positive in three of the four (75%) patients presenting no detectable antibodies in MAT, IgM ELISA, or slide agglutination test [70]. Moreover, the combination of the serological methods and PCR could enhance the sensitivity (93.1%–96.5%) in the first phase of leptospirosis [70].
The following generation of PCR technique, real-time PCR, has been applied to gain detection capability [57, 63]. Real-time PCR has faster turnaround time and more robust sensitivity and fidelity than conventional PCR [58, 61]. The single-tube assay with simultaneous detection prevents false-positive detection caused by amplicon contamination. Unlike conventional PCR, real-time PCR is a one-step assay with no requirement of downstream process such as gel electrophoresis and ethidium bromide staining, thus it reduces the risk of exposure to chemical hazards. In addition, this platform could be further applied to quantitate leptospiral burden in patient bodies, which may be useful to monitor response to antibiotic treatment, vaccine efficacy, and correlation of bacterial burden with disease severity [20, 80].
The SYBR green real-time PCRs targeting different genes, such as
Although SYBR green real-time PCR needs extra time for melting curve analysis, the cost of reagents is currently affordable and may compensate for the drawback. The melting peak is unique for each amplicon; therefore, it can be used to discriminate genome identity. Simultaneous pathogen detection and species classification is the advantage of SYBR green exceeding molecular probe real-time PCR. The combination of SYBR green-based detection and high-resolution melting (HRM) analysis becomes a novel and rapid diagnostic assay for leptospiral genotyping based on melting profiles [81, 82, 83]. This technique consists of amplification and detection of target genes, e.g.,
TaqMan probe real-time PCRs targeting unique genes of pathogenic serovars, such as
The prevalence of leptospirosis cases caused by intermediate
Additionally, the combination of
The transcript of constitutively expressed 16S rRNA gene is more abundant than its corresponding genomic DNA in viable bacteria; therefore, detection of the gene transcript should be more sensitive. The 16S rRNA gene transcripts of
Droplet digital PCR (ddPCR) is a novel nucleic acid quantification method that can be subsequently applied to TaqMan probe, SYBR green, or reverse transcription ddPCR [85, 86, 87, 88]. The reaction mixture is generated into water–oil emulsion nanosize particles that separate a single DNA template molecule into individual droplets. The positive detection refers to the fluorescent signal in each droplet representing single target copy per droplet. The direct quantification is estimated by the total count of positive droplets; therefore, the DNA standard curve is not required. Several publications reported the advantages of ddPCR in terms of sensitivity, accuracy, and reproducibility than real-time PCR to detect the causative agents of infectious diseases in clinical samples with very low level of targeted genomes [86, 88, 89, 90, 91, 92, 93]. However, current ddPCR systems compared with qPCR are very expensive, have low throughput, and have longer turnaround time to be used for routine diagnosis.
In theory, ddPCR might be useful for diagnosis of leptospirosis in the early phase because of low-abundance
The leptospirosis cases commonly present at hospitals in the rural areas. The rapid detection with less expensive and simpler methods such as loop-mediated isothermal amplification (LAMP) and isothermal recombinase polymerase amplification (RPA) assay have been determined for diagnosis of leptospirosis [78, 79, 94, 95, 96, 97]. Similar targets to PCR methods,
Early diagnosis of leptospirosis especially in severe cases is crucial for prompt and appropriate treatment to minimize morbidity and mortality. Nucleic acid detection tests are recommended for detection of