ABSTRACT.
Amoebic liver abscess (ALA) is the most common extra-intestinal complication of Entamoeba histolytica, accounting for 50,000 deaths annually, and is endemic in South Asia. Diagnosis based on microscopic examination is insensitive, and serological assays are not discerning of current infections in endemic settings with high exposure. For a rapid and confirmatory laboratory diagnosis of ALA, the performance of a polymerase chain reaction (PCR), quantitative real time PCR (qPCR), digital droplet PCR (ddPCR), and a loop-mediated isothermal amplification (LAMP) assay that detects E. histolytica DNA in liver abscess pus, and a lectin antigen detection ELISA were evaluated against clinical diagnosis (based on predefined criteria) as the gold standard. Owing to the lack of a laboratory gold standard, a Bayesian latent class analysis approach was also used to determine sensitivity and specificity of these assays. In the latent class analysis, qPCR and ddPCR showed the highest sensitivity (98% and 98.1%) and specificity (both 96.6%), and although clinical diagnosis had a comparable sensitivity to qPCR and ddPCR (95.2%), poorer specificity (64.3%) was seen. Kappa agreement analysis showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64–0.88) with PCR. Considering the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment needed, this would be the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA in the tertiary care laboratory setting in India, whereas further optimization of LAMP or antibody-based detection is required for use at smaller or secondary hospitals.
INTRODUCTION
The most common extra-intestinal complication caused by Entamoeba histolytica is amoebic liver abscess (ALA), accounting for 50,000 deaths annually and considered the fourth-leading cause of mortality and morbidity due to a parasitic infection.1 Although mortality rates have declined markedly in recent years, potentially attributable to early detection, there remains a significant risk of morbidity. Unlike pyogenic liver abscess (PLA), which has a global distribution, the burden of ALA is predominantly in low- to middle-income countries, with an annual incidence as high as 21 in 100,000 in Asia.2–4 A high index of suspicion is required for the diagnosis of ALA as it can emerge several years after infection and can present with features similar to those of PLA.4 Although highly sensitive imaging techniques such as ultrasonogram, computed tomography, and magnetic resonance imaging aid in early detection of liver abscesses of varied etiologies, they fail to differentiate ALA from PLA.2,5 Most often, a clinical diagnosis hinges on the history of socioeconomic status (with lack of safe drinking water) and alcoholism and the location and type of abscess (i.e., single abscess in the right lobe of the liver).6,7 A range of laboratory and imaging criteria such as leukocytosis, high random blood sugar, prolonged prothrombin time, low serum albumin, high alkaline phosphatase, hyperbilirubinemia, size and number of abscesses, abscess diameter, and volume of abscess cavity have been linked to clinical diagnosis, but they are nonspecific and cannot conclusively differentiate ALA from PLA and other space-occupying hepatic lesions, further highlighting the need for a microbiological diagnosis.5
Laboratory diagnosis of ALA includes demonstration of E. histolytica trophozoites in liver abscess aspirate, which is a challenge for even an experienced microscopist, with trophozoites observed in <10% of samples.8 Detection of the E. histolytica Gal/GalNAc lectin antigen in liver abscess pus is diagnostic and has also been used to detect circulating antigen in serum samples.9 Serological tests (IgG ELISA or indirect hemagglutination) have a significant advantage by avoiding invasive sampling; however, they can be misleading in endemic areas as they cannot distinguish an acute, ongoing infection from a prior infection or exposure.10 Recently, a point-of-collection (POC) lateral flow assay to detect IgG4 showed high specificity, but sensitivity was variable with geographic setting.11 Nucleic acid detection methods including polymerase chain reaction (PCR), quantitative real time PCR (qPCR), and loop-mediated isothermal amplification (LAMP), usually targeting the SSU rRNA gene, have been more reliable for diagnosis of ALA than antigen or antibody detection methods with high sensitivity and specificity.12–16 More recently, detection of cell-free DNA in serum has also been potentially useful for diagnosis of ALA, particularly because it does not require invasive liver aspiration.17
In this study, we carried out a comprehensive evaluation of molecular diagnostic methods for ALA comparing (LAMP), nested PCR, qPCR), and digital droplet PCR (ddPCR) for testing liver abscess aspirates from patients meeting clinical and radiological criteria. In the absence of a laboratory gold standard, these tests were compared with a clinical diagnosis and using Bayesian latent class analysis to yield valuable information on optimal diagnostics for ALA in an endemic country.
MATERIALS AND METHODS
Study subjects and sample collection.
All patients older than 15 years at the Christian Medical College Vellore, a tertiary healthcare center in India, with the following clinical and radiological inclusion criteria for evidence of liver abscess were included in this study between April 2011 and August 2012: fever, pain in the right hypochondrium, enlarged and/or tender liver with or without jaundice or intercostal tenderness, and single or multiple hypoechoic lesions in the liver by ultrasonography of the abdomen. Patients were then clinically categorized as “ALA,” “probable ALA,” and “PLA or other” by a physician prior to additional laboratory testing using the following criteria. Patients with a low-grade fever, right upper abdominal pain, intercostal tenderness, single large abscess in the right lobe of the liver on imaging, treated only with anti-amoebic therapy with a good response, with all tests for a PLA being negative with or without additional positive microbiological evidence for E. histolytica were defined as ALA cases. Patients with a single or multiple abscesses treated with antibiotics for both ALA and PLA with good response but no conclusive demonstrable microbiological evidence for either were defined as probable ALA cases. Patients with clinical evidence of a cholangitic sepsis or microbiological demonstration of an alternative causative organism from liver abscess pus were defined as PLA cases. Liver abscess aspiration was carried out under ultrasound guidance by an interventional radiologist and was performed for clinical purposes as judged by the admitting clinician for patient care or diagnosis and not solely for the purpose of this study. Blood and stool samples were also collected on enrollment in the study.
Laboratory methods.
After initial screening by microscopy and culture, pus and stool samples were stored at −80°C and serum was stored at −20°C until further testing. Laboratory staff members were blinded to the clinical diagnosis. Antigen-detection ELISA was performed on available liver aspirate samples, serum samples, and stool samples with the TechLab E. histolytica II kit (TechLab, Inc., Blacksburg, VA) according to the manufacturers’ instructions. DNA was extracted from the pus and stool samples with the QIAamp DNA Stool Minikit (Qiagen Inc, Valencia, CA). The LAMP assay was carried out using previously published primers targeting the SSU rRNA gene with a fluorescent detection reagent (Eiken Chemical Co., Ltd., Tokyo, Japan).18 Nested PCR was carried out with previously published primers also amplifying the SSU rRNA region with E. histolytica–specific primers (EH1 and EH2) in the second round, resulting in a 900-bp product.13 A subset of PCR products were sequenced with the BigDye Terminator ready reaction kit (Applied Biosystems, South San Francisco, CA) using the ABI PRISM 310 Genetic Analyser, and BLAST analysis was carried out to confirm the presence of E. histolytica. A Taqman-based qPCR with Platinum® qPCR SuperMix-UDG (Thermo Fisher Scientific, Walton, MA) amplifying the SSU rRNA gene with previously published primers/probe was carried out using the 7500 Fast Real-time PCR system (Applied Biosystems).19 Any sample with a cycle threshold (Ct) value <40 was considered positive for E. histolytica.
Digital droplet PCR was standardized using the same primers and probes as the qPCR using the Bio-Rad (Hercules, CA) ddPCR supermix for probes, which was subjected to droplet generation using a QX200™ Droplet Generator according to the manufacturer’s instructions (Bio-Rad). The droplets were then transferred to a 96-well PCR plate and heat-sealed with a foil cover, and amplification was carried out using a Veriti™ thermal cycler (Applied Biosystems) with the following cycling conditions: 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds, 60°C for 1 minute, and 98°C for 10 minutes. After amplification, the PCR plate was transferred to a QX200 Droplet Reader (Bio-Rad). All samples were tested in duplicate, and samples that had a low qPCR Ct value (Ct value ranging between 20 and 24) were tested in the ddPCR using both neat and 1:100 diluted DNA. All samples with >3 droplets were considered positive.20,21 Positive control DNA from E. histolytica strain HM-1:1MSS (obtained from Graham Clarke, London School of Tropical Medicine and Hygiene) was included in standardization of the molecular assays.
STATISTICAL ANALYSES
The results of the molecular diagnostic tests were analyzed by two approaches. First, the sensitivity and specificity of the four molecular diagnostic tests were calculated against a clinical diagnosis using STATA, Release 16.0 (StataCorp LLC, College Station, TX). Second, a Bayesian latent class analysis (BLCA) was applied to estimate the sensitivity and specificity of the four diagnostic tests with clinical diagnosis using the R-4.0.2 version.22 The sensitivity and specificity of the tests for indeterminate results (probable ALA cases) were assessed by considering them as both ALA negative and ALA positive. Patients with incomplete data were not included in the analysis. The agreement between the four molecular assays was estimated using Cohen’s kappa.23
RESULTS
Of the 57 cases recruited for this study, complete data and molecular test results were available for 54 cases and were included in the analysis (Figure 1). A majority of cases were males (88.8%), and the median (interquartile range) age at diagnosis was 45 (35–55) years. The mean duration of fever was 44 (1–365) days, with the duration of symptoms being greater than 2 weeks in 61% of cases. On examination, hepatomegaly was seen in 51 cases with a mean liver span of 3.1 (2–8) cm below the right costal margin. Right intercostal tenderness was seen in 92.5%, with a right pleural effusion in 68.5% of the cases. Peripheral leukocytosis, hyperbilirubinemia, and raised alkaline phosphatase levels were observed in 57.4%, 37%, and 70.3% of cases, respectively. On ultrasound imaging, 61% of patients had a single liver abscess. Three patients had a perforated liver abscess requiring surgical drainage, and 50 (85.1%) patients required percutaneous drainage.
Figure 1.
Flowchart showing study participant recruitment and eligibility. ALA = amoebic liver abscess; PLA = pyogenic liver abscess.
Of the 54 patients recruited for this study, based on clinical diagnosis 27 were classified as ALA, 14 were classified as PLA or others, and 13 as probable ALA (Supplemental Table 1). Among the PLA or others group, 12 patients were diagnosed as having PLA, one had a malignancy, and one had a tuberculous abscess of the liver. Out of 12 PLA cases, 11 aspirates were bacterial culture positive, with Klebsiella spp. being the most common pathogen isolated (4/11). Among the ALA and probable ALA cases, 3/40 were pus culture positive and grew methicillin-resistant Staphylococcus aureus, nonfermenting gram-negative bacteria, and Edwardsiella spp. Only one of the 27 aspirates from among the ALA cases was microscopy positive for E. histolytica trophozoites. When stored pus/aspirate samples were tested after enrollment with the TechLab E. histolytica ELISA, only two ALA cases along with two probable ALA cases and one of the PLA cases were positive. These results were not analyzed further owing to the low numbers detected.
Performance of nucleic acid detection–based laboratory tests.
Compared with clinical diagnosis as a gold standard, when probable ALA cases were considered ALA positive, LAMP assays had the highest sensitivity (100%), whereas both qPCR and ddPCR had high sensitivity (97.1%) and showed the highest specificity (65%) (Table 1). When probable ALA cases were considered, ALA negative, qPCR, and ddPCR had the highest sensitivity (64.7%) and specificity (75%) compared with clinical diagnosis as a gold standard. In the latent class analysis, irrespective of whether probable ALA cases were considered, ALA positive or negative, qPCR, and ddPCR showed the highest sensitivity (98–98.1%) and specificity (96.6%). When probable ALA cases were considered ALA positive, clinical diagnosis had a sensitivity comparable to that of qPCR and ddPCR (95.2%), but poorer specificity (64.3%) was seen. Kappa (agreement analysis; Supplemental Figure 1) showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64–0.88) with PCR.
Table 1.
Performance characteristics of LAMP, nested PCR, qPCR and ddPCR for diagnosis of Amoebic Liver Abscess Diagnostic test
| Considering “Probable ALA” Diagnosis as ALA Positive | Considering “Probable ALA” Diagnosis as ALA Negative | |||||||
|---|---|---|---|---|---|---|---|---|
| Sensitivity% (95% CI) | Specificity% (95% CI) | PPV % (95%CI) | NPV % (95%CI) | Sensitivity % (95% CI) |
Specificity % (95% CI) |
PPV % (95%CI) | NPV % (95%CI) | |
| Clinical Diagnosis as ‘gold standard’ | ||||||||
| Clinical diagnosis | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| LAMP | 100 (80.5–100.0) | 37.8 (22.5–55.2) | 42.5 (27.0–59.1) | 100 (76.8–100.0) | 58.8 (32.9–81.6) | 54.1 (36.9–70.5) | 37.0 (19.4–57.6) | 74.1 (53.7–88.9) |
| PCR | 87.1 (70.2–96.4) | 43.5 (23.2–65.5) | 67.5 (50.9–81.4) | 71.4 (41.9–91.6) | 64.5 (45.4–80.8) | 69.6 (47.1–86.8) | 74.1 (53.7–88.9) | 59.3 (38.8–77.6) |
| qPCR | 97.1 (84.7–99.9) | 65.0 (40.8–84.6) | 82.5 (67.2–92.7) | 92.9 (66.1–99.8) | 64.7 (46.5–80.3) | 75.0 (50.9–91.3) | 81.5 (61.9–93.7) | 55.6 (35.3–74.5) |
| ddPCR | 97.1 (84.7–99.9) | 65.0 (40.8–84.6) | 82.5 (67.2–92.7) | 92.9 (66.1–99.8) | 64.7 (46.5–80.3) | 75.0 (50.9–91.3) | 81.5 (61.9–93.7) | 55.6 (35.3–74.5) |
| Bayesian Latent Class Analysis (BLCA) | ||||||||
| Clinical diagnosis | 95.2 (85.4–99.4) | 64.3 (44.6–81.7) | 81.7 (68.9–91.3) | 89.1 (67.6–98.5) | 66.9 (51.9–80.7) | 73.3 (53.1–88.7) | 81.1 (63.8–92.1) | 57.5 (39.2–74.6) |
| LAMP | 50.0 (34.4–65.5) | 96.9 (85.3–99.9) | 96.4 (83.3–99.8) | 52.9 (38.3–69.2) | 46.8 (31.4–63.3) | 96.9 (85.0–99.9) | 96.2 (80.5–99.8) | 52.2 (36.0–68.4) |
| PCR | 75.4 (60.7–88.2) | 72.8 (54.4–88.2) | 82.6 (67.6–92.1) | 64.0 (44.1–81.7) | 75.5 (59.6–88.0) | 73.2 (54.5–88.7) | 82.7 (67.0–93.3) | 64.2 (44.4–81.9) |
| qPCR | 98.1 (89.7–99.9) | 96.6 (83.8–99.9) | 98.0 (89.4–99.9) | 96.8 (83.7–99.8) | 98.0 (88.9–99.9) | 96.6 (82.9–84.6) | 98.0 (88.6–99.9) | 96.6 (83.0–99.9) |
| ddPCR | 98.0 (90.5–99.9) | 96.6 (84.1–99.9) | 98.0 (89.9–99.9) |
96.8 (84.4–99.9) |
98.0 (88.9–99.9) | 96.8 (84.6–99.9) | 98.1 (90.0–99.9) |
96.6 (83.0–99.9) |
LAMP: Loop mediated isothermal amplification; PCR: Nested PCR;
qPCR: Real time PCR; ddPCR: Digital droplet PCR; PPV: Positive predictive value; NPV: Negative predictive value;
95% CI: Confidence interval (for clinical diagnosis gold standard) and Credible interval (for BLCA)
Testing of serum and stool.
Concurrent serum samples were available for all cases and were tested with the TechLab E. histolytica ELISA, but no positive samples were detected. Concurrent stool samples were available only for a subset of cases (seven ALA, four probable ALA, and four PLA/others), and among these, none were microscopy positive. The LAMP assay detected one positive sample in each of the three clinical categories, whereas nested PCR detected four positive samples among ALA cases and one each among the probable ALA and PLA and other cases.
DISCUSSSION
Amoebic liver abscess is suspected clinically in endemic areas in persons presenting with fever, abdominal pain, and liver tenderness. In this study, age, clinical presentation, duration of symptoms (>2 weeks), biochemical parameters, and radiological features were mostly comparable to previous reports from other parts of India.24–26 Hyperbilirubinemia (20.3%) and presence of coexistent cough and diarrhea (in 4.5% and 2.5% of cases, respectively) were lower than previously reported.27–29
We compared a range of molecular assays for laboratory diagnosis of ALA and showed that both qPCR and ddPCR performed with high sensitivity and specificity compared with a clinical diagnosis as gold standard and in the BLCA. Because of the identical results obtained from qPCR and ddPCR assays and taking into consideration the lower cost and relative ease of performance of qPCR as well as the wider availability of equipment needed (now further augmented by diagnostic facilities set up for the COVID-19 pandemic), this would be the most optimal assay for molecular diagnosis of ALA in the laboratory setting at the tertiary care level in India. Application of ddPCR for development of less-invasive assays to detect cell-free DNA in plasma has been explored by others.30,31 In our experience, the LAMP assay was most sensitive compared with clinical diagnosis but had a lower-than-expected specificity. The LAMP-based assays could be considered for use in low-resource settings for both laboratory and POC diagnostics.12,16,32 Improvements in the sensitivity and specificity of LAMP have been worked on by amplification of the signal detected, for example, with self-quenching probes and detection with lateral flow assay or lateral flow biosensors.33,34 Although other studies, including in India, have shown that the TechLab E. histolytica ELISA was useful for the detection of E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus, the reported sensitivity of 80% was reduced to 25% in samples collected after amoebicidal drugs were started.9,35 The possible reason for poor performance of this ELISA in our study could be prior use of metronidazole by the primary treating team.
Our study had its obvious limitation of suboptimal sample size but was an important first step toward evaluating multiple available molecular assays for confirmation of clinical diagnosis in an endemic setting. The other limitation that may have influenced the results of the tests is that a majority of the patients had received metronidazole empirically before aspiration of the abscess. We also did not evaluate any serological assays against the molecular tests. We did carry out a comprehensive evaluation of molecular assays, including ddPCR, for diagnosis of ALA in an endemic country setting to provide useful information on the utility of the various molecular assays. In tertiary care facilities in endemic countries such as India, where a large number of cases may be pretreated, the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment would make this the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA. Further optimization of LAMP and other potential POC detection from noninvasive samples, such as serum, is required for use at smaller or secondary hospitals.
Supplemental Materials
ACKNOWLEDGMENTS
We acknowledge the support of Ms. Selvi, the laboratory supervisor, and other technical staff for sample receipt and archival. We thank all the patients who consented to participate in the study. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.
Note: Supplemental materials appear at www.ajtmh.org.
Contributor Information
Sitara Swarna Rao Ajjampur, Email: sitararao@cmcvellore.ac.in.
Priscilla Rupali, Email: prisci@cmcvellore.ac.in.
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