Progress in the research on diagnosis and vaccines in amebiasis

Abstract

Entameba histolytica causes amebiasis, which includes both intestinal and extraintestinal amebiasis. E. histolytica causes 34 million to 50 million symptomatic cases of amebiasis worldwide every year, causing 40 thousand to 100 thousand deaths annually. E. histolytica, the pathogenic species of amebae is indistinguishable in its cyst and trophozoite stages from those of E. moshkovskii, a free-living ameba, and E. dispar, a non-invasive ameba, by microscopy, except in cases of invasive disease, where E. histolytica trophozoite may contain ingested red blood cells, but such a finding is rarely seen. This leads to a confusing scenario for the definite identification and differentiation of E. histolytica from E. moshkovskii and E. dispar by conventional microscopy, in the diagnosis of intestinal amebiasis. The advent of molecular methods such as multiplex PCR and real time PCR have facilitated a better and accurate diagnosis of E. histolytica, E. moshkovskii, and E. dispar in stool, urine, saliva, and other specimens. Multiplex PCR for the diagnosis of amebic liver abscess, using urine and saliva as clinical specimens, has been used, and the results have been encouraging. Real-time PCR is a new and a very attractive methodology for laboratory diagnosis of amebiasis, because of its characteristics that eliminate post-PCR analysis, leading to a shorter turnaround time. Microarray-based approaches represent an attractive diagnostic tool for the detection and identification of amebae in clinical and epidemiological investigations. Development of vaccines against amebiasis is still in its infancy. However, in recent years, progress has been made in the identification of possible vaccine candidates, the route of application, and the understanding of the immune response, which is required for protection against amebiasis. Thus, it is just a matter of time, and hopefully, amebiasis vaccine for human trials will be available in the next few years.

INTRODUCTION

Entameba histolytica the causative agent of amebiasis causes 34 million to 50 million symptomatic cases of amebiasis worldwide every year, causing 40 thousand to 100 thousand deaths annually.[1] E. histolytica, the pathogenic species of amebae is indistinguishable in its cyst and trophozoite stages from those of Entamoeba moshkovskii, a free-living ameba,[2] and Entamoeba dispar, a non-invasive ameba, by microscopy, except in cases of invasive disease, where E. histolytica trophozoite may contain ingested red blood cells,[3] but such a finding is rarely seen. This leads to a confusing scenario for the definite identification and differentiation of E. histolytica from E. moshkovskii and E. dispar by conventional microscopy, in the diagnosis of intestinal amebiasis.[2] The sensitivity of light microscopy is limited, at best, to only 60%.[2,3] The isoenzyme analysis of a cultured ameba allows the differentiation of E. histolytica from E. dispar. The isoenzyme analysis, however, takes a long time, usually between one to several weeks, to report the results and also requires special laboratory facilities, making it impractical for use in the routine diagnosis of intestinal amebiasis.

MOLECULAR DIAGNOSIS OF AMEBIASIS

To make a definitive diagnosis of amebiasis, stool culture followed by isoenzyme analysis enables the differentiation of E. histolytica from E. dispar. However, isoenzyme analysis requires one to several weeks to obtain the result and also special laboratory facilities are required; making it impractical for use in the routine diagnosis of intestinal amebiasis. Several Enzyme Linked Immunosorbent assay (ELISA) kits for antigen detection from the stool are available. These ELISA tests have a sensitivity approaching that of stool culture and can be performed rapidly. Antigen-based ELISA kits that are specific for E. histolytica use monoclonal antibodies against the Gal / GalNAc-specific lectin of E. histolytica (E. histolytica II; TechLab, Blacksburg, VA) or monoclonal antibodies against the serine-rich antigen of E. histolytica (Optimum S kit; Merlin Diagnostika, Bornheim-Hersel, Germany). Other ELISA kits for antigen detection include the Entameba CELISA PATH kit (Cellabs, Brookvale, Australia), which uses a monoclonal antibody specific for the lectin of E. histolytica, and the ProSpecT EIA (Remel Inc.; previously manufactured by Alexon-Trend, Inc., Sunnyvale, CA).[1–4]

The sensitivity and specificity of monoclonal antibody-based ELISA for the detection of E. histolytica specific antigen, in the stool specimen of intestinal amebiasis patients has been reported to be 85 and 90%, respectively.[4] The ELISA for antigen detection and nested PCR targeting of the 16S-like rRNA gene of E. histolytica has shown comparable sensitivities, 85 and 87%, respectively, when performed directly on fresh stool specimens.[5,6] The main limitation of the ELISA test is that it can specifically detect E. histolytica and E. dispar but not E. moshkovskii. To date E. moshkovskii in humans has been reported from North America, Italy, South Africa, Bangladesh, and India.[7–9] Our first report of E. moshkovskii was from India.[9] The presence of morphologically similar forms like E. dispar and E. moshkovskii has made the differentiation of the three species by PCR necessary, especially in countries like India where all the three species, E. histolytica, E. dispar, and recently E. moshkovskii have been reported to be prevalent.[10] The differential diagnosis of pathogenic E. histolytica from non-pathogenic E. dispar and E. moshkovskii will enable the clinicians to avoid unnecessary treatment with anti-amebic drugs, as colonization with E. dispar and E. moshkovskii does not warrant anti-amebic therapy.

Our center (JIPMER, Puducherry) was the first to develop and standardize multiplex nested PCR on stool samples for diagnosis of amebiasis. In a study from our center, PCR was positive in a total of 190 out of 202 stool specimens that were positive for E. histolytica / E. dispar / E. moshkovskii by microscopy and / or culture. All the 35 negative control stool samples that were negative for E. histolytica / E. dispar / E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). Furthermore, the result from the study showed that only 34.6% of the patient stool samples that were positive for E. histolytica / E. dispar / E. moshkovskii, by examination of stool by microscopy and / or culture, were actually positive for pathogenic E. histolytica and the majority of the stool samples remaining were positive for non-pathogenic E. dispar or E. moshkovskii, as demonstrated by the use of the nested multiplex PCR.[11] We also tried multiplex PCR for diagnosis of amebic liver abscess, using urine and saliva, as clinical specimens and found encouraging results.[12,13]

Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, which requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown toward the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. We reported for the first time the detection of E. histolytica DNA excreted in the urine, for the diagnosis of cases of amebic liver abscess (ALA). The PCR detected E. histolytica DNA in only four of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but DNA was detected in 17 out of 30 (56.7%) urine specimens collected after treatment with metronidazole. The present study for the first time showed that the kidney barrier in ALA patients was permeable to the E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in the urine, which could be detected by PCR. The study also showed that PCR, for detection of E. histolytica DNA in the urine of patients with ALA, can also be used as a prognostic marker, to assess the course of the disease, following therapy by metronidazole. The detection of E. histolytica DNA in the urine specimen of ALA patients provides a new approach for the diagnosis of ALA.[12]

Furthermore, we worked on the saliva sample, as saliva is an easily-accessible and a non-invasive clinical specimen, alternate to blood and liver pus. We detected E. histolytica DNA released in the saliva of ALA patients, by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole, but was positive in the saliva of eight (40%) of 20 ALA patients who were tested after therapy with metronidazole. This study, for the first time, demonstrated the release of E. histolytica DNA in the saliva of the ALA patients, by applying NM-PCR.[13]

In a recent study from Australia, 110 stool microscopy-positive fecal samples containing the Entamoeba species were subjected to PCR for confirmation of diagnosis of the Entamoeba species. The PCR products were detected in 89 (81%) samples, whereas, 21 (19%) samples were found to be negative by PCR assay. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively. Thus, only 4.5% of microscopy positive stool samples were actually positive for pathogenic E. histolytica. Another recent study from Tanzania in HIV positive individuals was conducted, in which stool samples were collected from 138 patients. Ten specimens were positive by microscopy, and PCR results showed that four were E. histolytica, one was E. dispar, and two were mixed infections with E. histolytica and E. dispar. Three were PCR negative, which could reflect inhibition of PCR by stool, DNA degradation or non-histolytica E. dispar / moshkovskii infections.[14,15]

Real-time PCR is a new and a very attractive methodology for laboratory diagnosis of infectious diseases, because of its characteristics that eliminate post-PCR analysis, leading to a shorter turnaround time. In addition, the closed reaction tube minimizes the chances of cross-contamination and the assay output is quantitative rather than qualitative. Therefore, the main advantage of this technique is that it can monitor the parasite load. Real time PCR has been employed for diagnosis of E. histolytica and E. dispar from DNA extracted from stool and liver abscess pus. For a single-plex, real-time, PCR detection of E. histolytica, Qvarnstrom et al., used TaqMan probes targeting the 18S rRNA gene, with the SYBR Green approach. Verweij et al., developed a multiplex qPCR assay for the detection of three different intestinal parasites E. histolytica, Giardia lamblia, and Cryptosporidium parvum. Their study showed 100% amplification of E. histolytica and G. lamblia DNA in microscopically positive isolates.[15] Later Haque et al., developed a multiplex real-time PCR assay for the detection of E. histolytica, G. lamblia, and C. parvum. The detection limit for the multiplex real-time PCR was one trophozoite of E. histolytica per extraction. The multiplex qPCR assay demonstrated 85% agreement with microscopy for E. histolytica.[15]

In a recent study from Spain, real-time PCR has been used for the detection and differentiation of E. histolytica and E. dispar infections in African or South American patients. Fecal samples from all the 130 subjects had apparently been found to contain E. histolytica / E. dispar cysts by microscopic examination. Using the real-time PCR, E. histolytica DNA was detected in fecal samples from only 10 (7.7%) of the immigrants, while E. dispar DNA was detected in the samples from another 117 (90.0%) of the subjects. The use of such a PCR in the routine investigation of patients found positive for E. histolytica / E. dispar cysts (by microscopy) is recommended, especially in non-endemic areas.

MICROARRAY IN DIAGNOSIS OF AMEBIASIS

One application that has revolutionized the postgenomic era is the development and use of microarray technology. DNA microarray is a newly developed technology, used for the detection of pathogens that are rapid and sensitive. Oligonucleotide microarrays have been successfully applied for the diagnosis of many pathogens in recent years. Microarray-based approaches represent an attractive diagnostic tool for the detection and identification of parasitic species in clinical and epidemiological investigations. The first oligonucleotide microarray developed for the parallel detection of E. histolytica, E. dispar, G. lamblia, and C. parvum in a single assay with high specificity and sensitivity, which was reported by Wang et al.[17] MacFarlane and Singh, using the DNA Microarray, carried out the first large-scale expression profiling of Entamoeba species / strains and opened the door to the investigation of genetic and expression differences, which may relate to parasite virulence.[18] However, functional studies need to be performed to confirm the roles of these genes in amebic pathogenesis. The main limitations of the microarray technique for diagnostic purpose are the costs, its robustness, and labor inputs.

AMEBIASIS VACCINE

E. histolytica is distributed worldwide and is generally associated with poor sanitary and socioeconomic conditions. As a majority of developing countries cannot afford improvements in sanitary condition, amebiasis is at present poorly controlled. High morbidity and mortality associated with invasive amebic infection, in spite of effective therapy, suggests that interventions designed to reduce or eliminate the disease are needed. As humans are the only relevant hosts for E. histolytica, an appropriate control program could potentially eradicate amebiasis. A number of ameba proteins have been tested as possible vaccine candidates and some of the protein molecules have been found to be effective in the animal model.[19] The two most promising vaccine candidates are the 25-kDa serine rich E. histolytica protein called SREHP and the 260-kDa galactose and N-acetyl galactosamine-inhibitable ameba lectin (Gal/GalNAc). Other candidate proteins are peroxiredoxins, lipophophosphoglycans, and cysteine proteinases.[20]

THE SERINE-RICH E. HISTOLYTICA PROTEIN

Serine-Rich E. histolytica Protein (SREHP) acts as a chemoattractant to E. histolytica trophozoites and mediates the binding of trophozoites of E. histolytica to the mamallian cells. Recombinant SREHP, as a SREHP / Maltose binding protein (MBP), fusion protein, has been tested in animal models. Gerbils were vaccinated intraperitoneally with recombinant SREHP / MBP and were later administered direct hepatic inoculation of virulent E. histolytica trophozoites.[21] In a total of three trials, 85% of the vaccinated gerbils were completely protected from developing amebic liver abscess. In addition, severe combined immunodeficiency (SCID) mice, passively immunized with antiserum to recombinant SREHP, were also protected from developing experimentally induced amebic liver abscess, suggesting that mainly antibodies were responsible for the observed protection. The safety and immunogenicity of recombinant SREHP has been well-documented in African green monkeys.[22]

THE N-ACETYLGALACTOSAMINE-INHIBITABLE E. HISTOLYTICA LECTIN (GAL / GALNAC)

The lectin plays a key role in the pathogenesis of amebiasis, as it mediates adherence of trophozoites to colonic cells. The purified native Gal / GalNAc lectin has been used to vaccinate gerbils to protect them against amebic liver abscess. Although the vaccination has been protective in 66% of the animals, in the remaining there has been evidence of a significant increase in liver abscess size, suggesting that the immune response to the lectin could also exacerbate the disease.[23] Thus use of Gal / Gal NAc lectin as possible vaccine candidates is debatable.

OTHER VACCINE CANDIDATES

E. histolytica contains a 29-kDa cysteine-rich protein, which constitutes a peroxiredoxin that is able to inactivate hydroxyperoxide in host tissues. In a study, the intraperitoneal application of recombinantly expressed peroxiredoxin in gerbils revealed a systemic IgG response against the protein, and partial protection (54%) against intrahepatic challenge with E. histolytica trophozoites.[24] However, its safety has not been evaluated in primates.

Similarly lipophosphoglycans (LPGs) were found only in the pathogenic strains of E. histolytica. Monoconal antibodies were raised against it and were passively transferred in SCID mice. These SCID mice demonstrated a high degree of protection against amebic liver abscess development.[25] However, production of amebic LPGs for large scale vaccination trials was a major challenge, as these molecules were extensively modified with complex linear glycan chains attached to phosphoserine residues in the polypeptide backbone, and to date, all efforts to form recombinant LPG have failed.

ORAL AND INTRANASAL VACCINES

Amebiasis is associated with oro-fecal contamination, thus oral vaccines are also in the pipeline. In one such study attenuated Yersinia enterocolitica were generated, which upon invasion by Peyers patches, were able to release various portions of the 170-kDa lectin. Oral application of vaccines in rodents elicited a substantial IgA response to the amebic antigens. In addition, orally immunized gerbils were partially protected from amebic liver abscess formation after intrahepatic challenge with virulent E. histolytica trophozoites.[26] However, the degree of protection was much weaker in orally immunized animals compared to those vaccinated intraperitoneally. Intranasal purified native Gal / GalNAc lectin was applied in a C3H mouse model. The mice were given the intracecal challenge with E. histolytica trophozoites and 100% mice were protected from amebic colitis.[27]

SUMMARY

Advent of molecular methods such as multiplex PCR and real time PCR have facilitated better and accurate diagnosis of E. histolytica, E. moshkovskii, and E. dispar. Development of vaccines against amebiasis is still in its infancy. However, in recent years, progress has been made in the identification of possible vaccine candidates, the route of application, and the understanding of the immune response that is required for protection against amebiasis. Thus, it is just a matter of time before amebiasis vaccine for human trials will be available; possibly in next few years.