Evaluation of Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Acanthamoeba Keratitis

Background: The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%). The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings     

Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Malaria Infections in an Area of Endemicity in Thailand

The loop-mediated isothermal amplification (LAMP) method, developed by our group for diagnosis of four human malaria parasites, was evaluated on a large scale at a remote clinic in Thailand where malaria is endemic. A total of 899 febrile patients were analyzed in this study. LAMP was first evaluated in 219 patients, and the result was compared to those of two histidine-rich protein (HRP)-2 rapid diagnostic tests (RDTs) and microscopy as a gold standard. LAMP DNA extraction was conducted by a simple boiling method, and the test results were assessed visually. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 95.7%, 100%, 100%, and 98%, respectively, for LAMP and 98.6%, 98%, 95.8%, and 99.3%, respectively, for RDTs. Since RDT-positive results were based on one out of two RDTs, the sensitivity of RDTs was slightly higher than that of LAMP. However, LAMP tended to be more specific than RDTs. LAMP next was evaluated in 680 patients, and the result was compared to that of microscopy as a gold standard. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of LAMP were 88.9%, 96.9%, 92.2%, 95.5%, and 94.6%, respectively. Nested PCR was used to confirm the discrepant results. Malaria LAMP in a remote clinic in Thailand achieved an acceptable result, indicating that LAMP malaria diagnosis is feasible in a field setting with limited technical resources. Additionally, the rapid boiling method for extracting DNA from dried blood spots proved to be simple, fast, and suitable for use in the field.     

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis.Entamoeba histolytica is the etiologic agent of human amebiasis, which causes an estimated 40 to 50 million cases of dysentery and liver abscess and up to 100,000 deaths each year, mainly in tropical and subtropical countries (6). The detection of amebiasis in developing countries is currently dependent on microscopic examination of stool samples from patients. However, this system is at best only 10 to 60% sensitive (9, 10, 14, 22) and is incapable of distinguishing between the pathogenic species E. histolytica, the nonpathogenic species Entamoeba dispar, and the amphizoic amoeba Entamoeba moshkovskii, which infects humans sporadically (5, 7, 8). The lack of early and accurate diagnosis is a critical obstacle to clinical management of E. histolytica infection globally. Therefore, an accurate but feasible method that can be easily used in areas where amebiasis is endemic is clearly needed to confirm the identification of E. histolytica.Several new diagnostic approaches based on the detection of an E. histolytica-specific antigen and DNA had been established recently (22). The TechLab Entamoeba histolytica II kit is the only commercially available antigen test kit for specific detection of E. histolytica in feces. However, since storage or the use of preservatives destroys the antigen, it is recommended for use exclusively with fresh stool samples (21, 22). Several PCR-based assays for E. histolytica identification have subsequently been published, including single-tube multiplex reactions and nested PCR (12). The results suggest that PCR should be useful as a reference test for sensitive differentiation of E. histolytica and E. dispar (7) and could replace the TechLab enzyme-linked immunosorbent assay in areas where the pathogen occurs less frequently (23). However, the PCR assay commonly requires electrophoresis to detect the amplicons, leading to relatively high expense, including labor costs, and long turnaround time. Real-time PCR assays have also been applied to detect and quantify pathogens by continuously monitoring the amplicon formation with time (22). Nevertheless, such assays require an expensive thermal cycler with a fluorescence detector.The loop-mediated isothermal amplification (LAMP) assay was originally developed by Mori et al. (17), Nagamine et al. (18), and Notomi et al. (19) (Eiken Chemical Co., Ltd., Japan), using a set of two specifically designed inner primers and two outer primers that recognize six distinct regions of the targeted DNA. Since this reaction is performed under isothermal conditions, simple incubators, such as a water bath or heat block, are adequate for the DNA amplification. Considering these advantages, the LAMP assay could become a valuable diagnostic tool in developing countries or hospital laboratories. Therefore, the aim of this study was to develop a sensitive and specific LAMP-based method to detect E. histolytica and to evaluate this method by comparing it to a conventional nested PCR assay with clinical fecal samples.     

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Subgroup J Avian Leukosis Virus

Infection of breeder flocks in China with subgroup J avian leukosis virus (ALV-J) has increased recently. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of ALV-J from culture isolates and clinical samples. The ALV-J-specific LAMP assay efficiently amplified the target gene within 45 min at 63°C using only a simple laboratory water bath. To determine the specificity of the LAMP assay, various subgroup ALVs and other related viruses were detected. A ladder pattern on gel electrophoresis was observed for ALV-J isolates but not for other viruses. To evaluate the sensitivities of the LAMP assay and conventional PCR, the NX0101 isolate plasmid DNA was amplified by them. The detection limit of the LAMP assay was 5 target gene copies/reaction, which was up to 20 times higher than that of conventional PCR. To evaluate the application of the LAMP assay for detection of ALV-J in clinical samples, 49 samples suspected of ALV infection from breeder flocks were tested by the LAMP assay and PCR. Moreover, virus isolation from these samples was also performed using cell culture. The positive-sample ratios were 21/49 (43%) by conventional PCR, 26/49 (53%) by the LAMP assay, and 19/46 (41%) by virus isolation. Additionally, a positive LAMP reaction can be visually ascertained by the observation of turbidity or a color change after addition of SYBR green I dye. Consequently, the LAMP assay is a simple, rapid, and sensitive diagnostic method and can potentially be developed for rapid detection of ALV-J infection in the field.Avian leukosis viruses (ALVs), which belong to the Alpharetrovirus genus of the family Retroviridae, can induce transmissible benign and malignant neoplasms in poultry (13, 24). ALVs are divided into subgroups based primarily on virus neutralization, their host range, and viral interference patterns (4, 5, 13). The envelope glycoprotein (gp85) is responsible for subgroup specificity. In general, the endogenous nonpathogenic subgroup E viruses are present in nearly all chicken lines (13, 26). Subgroup C and D viruses are rarely seen in the field. The exogenous viruses (subgroups A, B, and J) mainly induce lymphoid leukosis (LL) and myeloid leukosis (ML) in field flocks (13). In the late 1980s, the prototype strain of subgroup J avian leukosis virus (ALV-J), HPRS-103, was first isolated from commercial meat-type chickens in the United Kingdom (22). ALV-J infection subsequently caused heavy losses worldwide in broiler breeder stocks. Many strains of ALV-J have been reported worldwide in the past 20 years (2, 11, 30). Sequence analysis has demonstrated that the gp85 genes of subgroups A to E are closely related, sharing around 85% nucleotide sequence identity with each other. However, the HPRS-103 gp85 gene shares only 40% identity with members of other subgroups. The gp85 genes of endogenous avian retroviral sequence (EAV-HP) elements share over 97% identity with that of ALV-J; thus, it was hypothesized that the new avian pathogen may be a result of recombination between exogenous and endogenous retroviruses (1, 4, 5, 25).ALV-A, ALV-B, and ALV-J are the subgroups most dangerous to the poultry industry (13). No treatment exists for the exogenous viral infection. Furthermore, no commercial vaccine is available, so eradication measures are carried out by frequent detection of exogenous ALVs (27). The emergence of ALV-J forced some poultry breeders to formulate new plans to control ALV-J (7, 14, 15). Since ALV-J is spread by both vertical and horizontal transmission. The only method for blocking the vertical transmission of ALV-J is the early elimination of infected chickens (6, 9). China has the second largest broiler industry in the world at present. However, ALV-J-induced ML in China was not officially documented until 1999 (11). ALV-J isolates have subsequently been obtained from broiler breeders and layer chickens in most parts of China (11, 30).PCR and real-time quantitative PCR methods were developed for detection of ALV-J (16, 17, 26, 27). However, PCR requires considerable operator skills and expensive equipment; thus, its application is limited in the field. Virus isolation using CEF or DF-1 cell culture takes more than 7 days for an accurate final result to be obtained, as does the immunofluorescence assay (IFA) specific for ALV-J. An enzyme-linked immunosorbent assay (ELISA) kit designed to detect the viral group-specific antigen (gsa; p27) is available. In China, a few large poultry breeders are trying to establish exogenous ALV-free poultry flocks, which were evaluated by ELISA detection. However, ELISA may lead to a high rate of false-positive results due to the p27 expression of some endogenous retroviruses. Thus, ELISA is not reliable for the detection of purely exogenous ALVs (10). A simple, rapid, and sensitive diagnostic method for detecting ALV-J needs to be developed.The loop-mediated isothermal amplification (LAMP) method was developed by Notomi et al. (20). This novel technique generally requires isothermal conditions and four different primers for DNA amplification (19, 20) and has been applied to the detection of several pathogens (3, 8, 12, 21, 23). The LAMP reaction requires 30 to 60 min and can be performed at a single temperature ranging from 60 to 65°C. LAMP does not require the DNA denaturation, annealing, and extension PCR cycles (19, 20). In addition, the results can be ascertained easily by the naked eye (3, 19). In this study, we developed a LAMP assay for simple, rapid, and sensitive detection of ALV-J and compared the sensitivity of the LAMP assay with those of conventional PCR and virus isolation.     

Development of a Loop-Mediated Isothermal Amplification Assay Targeting the mpb64 Gene for Diagnosis of Intraocular Tuberculosis

A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings.     

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

Aspergillus fumigatus is a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection of A. fumigatus infection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection of A. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatus strains, including 5 species of the Aspergillus genus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10² copies for the same target. Clinical samples from a total of 69 patients with probable IA (n = 14) and possible IA (n = 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection of A. fumigatus in clinical testing has been developed.     

Loop-Mediated Isothermal Amplification Method for Differentiation and Rapid Detection of Taenia Species

Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.     

Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

Rapid and accurate tests are currently needed to identify individuals with high levels of Loa loa microfilaria (mf), so that these individuals may be excluded from mass ivermectin administration campaigns against onchocerciasis and lymphatic filariasis being conducted in areas where Onchocerca volvulus, Wuchereria bancrofti, and L. loa are coendemic. To address this need, colorimetric loop-mediated isothermal amplification (LAMP) assays targeting the L. loa-specific gene sequences LLMF72 and LLMF342 were developed for the detection and quantification of L. loa microfilaremia. Both LAMP assays were highly specific (100%) for L. loa infection compared to the absence of infection or infection with related filarial pathogens. The LLMF72-based LAMP assay showed greater analytic sensitivity (limit of detection, 0.1 pg/ml of genomic DNA [gDNA] and/or 5 mf/ml) than the LLMF342-based LAMP assay (10 pg/ml of gDNA and/or 50 mf/ml), and its analytic sensitivity was similar to that of LLMF72-based quantitative PCR (qPCR). A high level of correlation was observed between microfilaria counts as determined by LLMF72-based qPCR and time to positivity by the LAMP assay, and performance measures of sensitivity, specificity, and positive and negative predictive values were similar for both assays when applied to field-collected clinical samples. By simply varying the run time, the LAMP assay was able to accurately distinguish individuals at risk for serious adverse events (SAEs) after exposure to ivermectin, using thresholds of >5,000 mf/ml and >30,000 mf/ml as indicators of increasing levels of risk. In summary, LLMF72 LAMP represents a new molecular diagnostic tool that is readily applicable as a point-of-care method for L. loa microfilarial detection and quantification in resource-limited countries where L. loa infection is endemic.     

Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia

Pneumonia due to Pneumocystis jirovecii (PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258–261, doi:http://dx.doi.org/10.1016/j.mimet.2008.06.009), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category, Pneumocystis jirovecii DNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detect Pneumocystis jirovecii DNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden.     

Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid and Inexpensive Detection of Cytomegalovirus DNA in Vitreous Specimens from Suspected Cases of Viral Retinitis

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of cytomegalovirus (CMV) was developed and evaluated. The LAMP assay specifically amplified only CMV DNA, and no cross-reactivity with the DNA of herpes simplex virus type 1, varicella-zoster virus, adenovirus, Aspergillus flavus, or Staphylococcus aureus was observed. The sequences of the LAMP assay-positive CMV products were perfectly (100%) matched with the CMV sequence deposited in the GenBank database. The sensitivity of the LAMP assay was found to be 10 copies/μl of CMV DNA. Vitreous samples from 40 patients with suspected retinitis were subjected to LAMP and real-time PCR for the detection of CMV. Of 40 patients with suspected viral retinitis, 10 tested positive for CMV by the real-time PCR and LAMP assays. A 100% concordance was observed between the results of the two methods. The LAMP assay is a rapid, highly specific, and sensitive method for the diagnosis of retinitis caused by CMV.Viral retinitis is commonly caused by herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), and occasionally, Epstein-Barr virus (EBV) (7). In patients with atypical features and in the early stages of ocular manifestations, clinical differentiation between cases of retinitis associated with CMV and other herpesvirus infections is often difficult (6). The differentiation of CMV retinitis from HSV and VZV retinitis is very important early in the course of the disease, as the therapeutic agent to be used for treatment differs from virus to virus (7). Conventional methods for the diagnosis of viral retinitis include the detection of viral antigen and virus isolation from intraocular specimens (2). These tests have been shown to have low sensitivities for the detection of viruses and are not currently recommended for use for the diagnosis of viral retinitis (2).PCR has proved to be of great utility for the diagnosis of viral retinitis (3, 4, 5, 6, 8). However, owing to the expensive systems required, PCR is still not a very common diagnostic test. Notomi et al. have reported on a novel nucleic acid amplification assay termed the loop-mediated isothermal amplification (LAMP) assay (10). The assay amplifies the DNA under isothermal conditions (63 to 65°C) with high degrees of specificity, efficiency, and speed. The assay can be conducted in the laboratory in a water bath or heating block (10). Thus, the thermal cycling needs of a PCR are avoided. The assay can be used for the rapid detection of pathogens in peripheral health care settings in developing countries. The present study describes the development and evaluation of a simple and cost-effective LAMP assay for the rapid detection of CMV DNA in patients with viral retinitis.     

Evaluation of Loop-Mediated Isothermal Amplification Assay for Detection and Typing of Human Papilloma Virus 16 and 18 from Endocervical Samples

Background: Many human papillomavirus (HPV) types are associated with cervical cancer (CC). Therefore, HPV genotyping has both clinical and epidemiological importance. HPV 16 and 18 are two principal high-risk types responsible for more than 70% of all CC cases. Although several commercial and non-commercial genotyping assays are available, there is a need for a cost-effective and sensitive genotyping method for low- and middle-income countries. Methods: The study was aimed at evaluation of loop-mediated isothermal amplification (LAMP) assay for HPV genotyping in cervical samples. A total of six primer sets for each HPV type were selected for the assay. The LAMP assay was standardised and validated with HPV control panel. Cervical biopsies were subjected to nested multiplex polymerase chain reaction (NM-PCR; as a part of routine diagnostic workup) and LAMP (HPV 16 and 18) simultaneously. Results: A total of 225 clinical samples were processed during the study period. The sensitivity of the assay was determined using the 10-fold dilutions of positive controls. Both the HPV 16-LAMP and HPV 18-LAMP assays were shown to detect as low as 10 viral copies per reaction, which is similar to that of NM-PCR. The LAMP assay had a good agreement (new cases; 92%, post-chemoradiotherapy [post-CRT]; 89.1%) with NM-PCR for the detection of both HPV 16 and 18. As compared to histology (new cases; 79.8%, post-CRT; 51.3%), LAMP had better agreement with NM-PCR for detection of HPV from post-CRT cases. Conclusions: We evaluated the LAMP assay for simultaneous detection and typing of HPV 16 and 18. The assay had good agreement with NM-PCR for detection of both HPV 16 and 18. The LAMP assay is a promising tool for HPV genotyping along with routine cervical cytology, especially in resource-constrained settings.     

Clinical Evaluation of the BDProbeTec Strand Displacement Amplification Assay for Rapid Diagnosis of Tuberculosis

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96.2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.     

Development of a Sensitive Loop-Mediated Isothermal Amplification Assay That Provides Specimen-to-Result Diagnosis of Respiratory Syncytial Virus Infection in 30 Minutes

Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (T_m). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.     

Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.     

Prospective Evaluation of Commercial Antibody-Based Rapid Tests in Combination with a Loop-Mediated Isothermal Amplification PCR Assay for Detection of Orientia tsutsugamushi during the Acute Phase of Scrub Typhus Infection

Samples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection of Orientia tsutsugamushi IgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity.     

Development and Clinical Evaluation of sdaA Loop-Mediated Isothermal Amplification Assay for Detection of Mycobacterium tuberculosis with an Approach To Prevent Carryover Contamination

A rapid and sensitive loop-mediated isothermal amplification assay for the sdaA gene of Mycobacterium tuberculosis was developed using a dUTP-uracil-N-glycosylase (dUTP-UNG) strategy to prevent carryover contamination. Evaluation of the assay using clinical specimens (n = 648) showed high specificity (97.2%) and sensitivity (100%), demonstrating its potential as a diagnostic test for tuberculosis, especially in resource-limited settings.