Balamuthia mandrillaris: An opportunistic, free-living ameba - An updated review.

Balamuthia mandrillaris is an opportunistic, free-living ameba that is pathogenic to humans. It has a worldwide distribution but is mainly detected in warmer regions. Balamuthia infections are rare but have been reported in both immunocompetent and immunocompromised individuals of all ages. B. mandrillaris can enter through wounds on the skin or the nose and cause cutaneous lesions and the usually fatal Balamuthia amebic encephalitis (BAE). Infection usually spreads from the lungs or through nerve fibers, and attacks the central nervous system, forming granulomatous lesions and necrosis in the brain. Balamuthia infection is usually chronic, and patients initially present with nonspecific symptoms, including headache, nausea, myalgia, and low-grade fever. As the disease progresses, the patient becomes paralyzed and comatose, often leading to death. Lack of knowledge of predisposing factors, specific treatment, and standardized detection tools have resulted in a nearly cent percent fatality rate. Although only about 200 cases have been reported worldwide since its characterization in the 1990s, the number of reported cases has increased over the years. BAE is an emerging disease and a major health concern. Few patients have survived Balamuthia infections with antimicrobial treatment that has largely been empirical. Early diagnosis is the key and requires familiarity with the disease and a high degree of suspicion on the part of the diagnostician. There are currently no specific treatment and prevention recommendations. This review highlights our current understanding of B. mandrillaris in terms of its pathogenicity, genomics, and novel diagnostic and therapeutic approaches against BAE infections.

Narciclasine improves outcome in sepsis among neonatal rats via inhibition of calprotectin and alleviating inflammatory responses.

Sepsis is associated with exacerbated inflammatory response which subsequently results in multiple organ dysfunction. Sepsis accounts for high mortality and morbidity among newborns worldwide. Narciclasine is a plant alkaloid which has shown to possess anti-inflammatory properties. In this study we investigated the effect and mechanism of action of narciclasine in neonatal sepsis rat models. The excessive release of S100A8/A9 or calprotectin in neonatal sepsis could be detrimental as it could exacerbate the inflammatory responses. We found that narciclasine significantly reduced the plasma levels of S100A8/A9 and also suppressed its expression in the liver and lung. The systemic and local bacterial load was also reduced in the narciclasine treated rats. The systemic and local production of pro-inflammatory cytokines in plasma and organs (liver and lungs) was significantly reduced in the narciclasine treated rats. The histopathological studies showed that narciclasine prevents the organ damage associated with sepsis and improved the survival of neonatal rats. Sepsis increased the phosphorylated NF-κß p65 protein expression in the liver. Narciclasine suppressed the phosphorylation of NF-κß p65 and the degradation of NF-κß inhibitory protein alpha. It could also suppress the expression of adaptor proteins of the toll like receptor signaling pathway viz., myeloid differentiation factor 88 (MyD88), Interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6). These results suggest that narciclasine protects against sepsis in neonatal rats through the inhibition of calprotectin, pro-inflammatory cytokines and suppression of NF-κß signaling pathway.

Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus: A preliminary report from south India.

Background & objectives: Although there are reports of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) across the globe, there is a lack of reliable data on hVISA in India. The present study was undertaken to determine the rate of hVISA among the methicillin-resistant Staphylococcus aureus (MRSA) isolates, and to compare the brain heart infusion agar with vancomycin 4 µg/ml (BHIV4) method with population analysis profile-area under the curve (PAP-AUC) method for the detection of hVISA and to study the distribution of mobile genetic element that carries methicillin-resistance gene SCCmec (Staphylococcal cassette chromosome mec) types among these isolates. Methods: BHIV4 and PAP-AUC methods were employed to detect hVISA among 500 clinical isolates of MRSA. SCCmec typing of these isolates was performed by multiplex polymerase chain reaction. The clinical presentation, treatment with vancomycin and outcome was documented for patients with hVISA. Results: The rate of hVISA was 12.4 per cent by PAP-AUC method. Sensitivity, specificity, PPV, NPV and kappa agreement of BHIV4 with PAP-AUC was 58.06, 93.15, 54.55, 94.01 per cent and 0.498, respectively. The isolation of hVISA was significantly (P<0.01) higher in patients admitted to intensive care units and wards than in patients attending the outpatient departments. Only 38 per cent of the patients received vancomycin as therapy. Majority of the hVISA isolates carried SCCmec type V or IV. Interpretation & conclusions: The rate of hVISA isolation in our study was 12.4 per cent. The sensitivity of the BHIV4 screening test was low, and was in moderate agreement with PAP-AUC test. SCCmec type V was the predominant type seen in half of the isolates. More studies need to be done in different parts of the country on a large number of isolates to confirm our findings.

Severe Blastocystis subtype 3 infection in a patient with colorectal cancer.

Colorectal cancer (CRC), more of lifestyle-related disorder, is one of the deadliest types of cancer across the globe. Nevertheless, infectious agents could be responsible for 20% of cancer. Recent findings have indicated the association of Blastocystis in CRC and recommend routine screening for Blastocystis. Herein, we describe a case of CRC with severe Blastocystis infection.

Multiple parasitic and viral infections in a patient living with HIV/AIDS on antiretroviral therapy.

Patients with human immunodeficiency virus (HIV) infection are more prone for gastrointestinal infections causing diarrhoea, particularly with parasites. Parasitic infections have been regularly reported in such patients. A female patient confirmed positive for HIV 1 on antiretroviral therapy came with complaints of chronic diarrhoea for the past 7 months. Her initial CD4 count was 89 cells/µl of blood, and antibodies to cytomegalovirus and herpes simplex virus 1 and 2 virus were found to be positive in the patient's serum, but there was no HIV-associated retinopathy. Her stool examination showed decorticated fertilised eggs of Ascaris lumbricoides, cysts of Blastocystis sp. and Entamoeba species in the unconcentrated sample and oocysts of Cystoisospora species, egg of Schistosoma haematobium and eggs of Trichuris trichiura in the concentrated. The patient responded well to cotrimoxazole and albendazole, and repeat samples were negative for all these parasites.

In-silico prediction and modeling of the Entamoeba histolytica proteins: Serine-rich Entamoeba histolytica protein and 29 kDa Cysteine-rich protease.

BACKGROUND: Amoebiasis is the third most common parasitic cause of morbidity and mortality, particularly in countries with poor hygienic settings. There exists an ambiguity in the diagnosis of amoebiasis, and hence there arises a necessity for a better diagnostic approach. Serine-rich Entamoeba histolyticaprotein (SREHP), peroxiredoxin and Gal/GalNAc lectin are pivotal in E. histolyticavirulence and are extensively studied as diagnostic and vaccine targets. For elucidating the cellular function of these proteins, details regarding their respective quaternary structures are essential. However, studies in this aspect are scant. Hence, this study was carried out to predict the structure of these target proteins and characterize them structurally as well as functionally using appropriate in-silicomethods. METHODS: The amino acid sequences of the proteins were retrieved from National Centre for Biotechnology Information database and aligned using ClustalW. Bioinformatic tools were employed in the secondary structure and tertiary structure prediction. The predicted structure was validated, and final refinement was carried out. RESULTS: The protein structures predicted by i-TASSER were found to be more accurate than Phyre2 based on the validation using SAVES server. The prediction suggests SREHP to be an extracellular protein, peroxiredoxin a peripheral membrane protein while Gal/GalNAc lectin was found to be a cell-wall protein. Signal peptides were found in the amino-acid sequences of SREHP and Gal/GalNAc lectin, whereas they were not present in the peroxiredoxin sequence. Gal/GalNAc lectin showed better antigenicity than the other two proteins studied. All the three proteins exhibited similarity in their structures and were mostly composed of loops. DISCUSSION: The structures of SREHP and peroxiredoxin were predicted successfully, while the structure of Gal/GalNAc lectin could not be predicted as it was a complex protein composed of sub-units. Also, this protein showed less similarity with the available structural homologs. The quaternary structures of SREHP and peroxiredoxin predicted from this study would provide better structural and functional insights into these proteins and may aid in development of newer diagnostic assays or enhancement of the available treatment modalities.

A Stratified Analysis of Clinical Manifestations and Different Diagnostic Methods of Neurocysticercosis-Suspected Tamilian Population Residing in and Around Puducherry.

INTRODUCTION: Human beings are accidental hosts for Cysticercus cellulosae showing varied clinical manifestations based on the site harbored by the parasitic cyst because of which disease profile remains unexplored at large. Besides this, diagnosis of the disease with a single conventional method is problematic due to lack of specificity and sensitivity. AIM: To assess the varied clinical manifestations and stratify based on imaging and serological methods for diagnosis of Neurocysticercosis (NCC) in our study population. MATERIALS AND METHODS: A hospital-based study was carried out at Jawaharlal Institute of Post-Graduate Medical Education and Research (JIPMER), the tertiary care centre caters patients from Puducherry and surrounding regions of Tamil Nadu. This is a cross-sectional analysis of clinically and radiologically suspected cases of NCC (n=119) for a period of three years (2012 to 2015). The collection of detailed clinical history and imaging findings (MRI or CT) along with the lifestyle parameters was done after obtaining informed consent. Enzyme-Linked Immune-Electro Transfer Blot (EITB) was carried out for the samples collected from study subjects. RESULTS: Based on dietary and environmental factors non-vegetarians, pork eaters, raw vegetable consumers and open-field defecation showed significant seropositivity. The clinical manifestations like seizures, blurring of vision and chronic headache with nausea followed by neck pain, cognitive deficits and movement disorder have higher seropositivity respectively. Generalized seizures were found to be more than focal seizures. While comparing the imaging and serological tests for NCC diagnosis, the positivity rate was 46.2% considering positive by both methods; but 18.5% of sero-positive cases were imaging negative, and 16% of the sero-negative cases were imaging positive. The study showed a predominance of multiple cysts (62%) in cases with cystic lesions. CONCLUSION: This study is first of its kind in associating varied and less commonly explored clinical manifestations with two different diagnostic measures in practice and its importance among our study settings. These manifestations must be considered as strong disease entities of NCC, which has to be suggested for differential diagnosis, and cannot be left ignored. Combinatorial diagnostic methods like serology and imaging techniques should be followed in diagnosis and assessing the disease burden.

Detection of Cryptosporidium in stool samples of immunocompromised patients.

INTRODUCTION: Cryptosporidium species is the most common opportunistic enteric parasite encountered in the immunocompromised patients. Considering the need to diagnose them early relies mostly on rapid tests such as antigen detection by immunochromatographic test (ICT), ELISA, and microscopy. However, the sensitivity and specificity varies with different methods and different kits used. This study was conducted to determine the intestinal parasitic profile in immunocompromised patients and to assess the diagnostic accuracy of the ICT using ImmunoCard STAT kit in detecting Cryptosporidium spp. MATERIALS AND METHODS: The patients in this study were divided into two groups: one group was immunocompromised patients (n = 73) and the other was nonimmunocompromised individuals (n = 73). Stool microscopy, ICT, and polymerase chain reaction (PCR) were carried out for all stool samples. RESULTS: Totally, 4 (5.4%) of 73 patients of the study group were positive for Cryptosporidium. The species detected were Cryptosporidium parvum and Cryptosporidium hominis. PCR was taken as gold standard in the current study. PCR detected Cryptosporidium in four samples while ICT in two samples and microscopy in one sample. CONCLUSION: Cryptosporidium was found to be the most common enteric parasite in the immunocompromised patients studied, followed by Cystoisospora, Entamoeba histolytica, and Strongyloides stercoralis. Although the ICT is a rapid test, it was less sensitive and more expensive in comparison to the PCR; hence, its utility appears to be limited in our setting.

Management of granulomatous amebic encephalitis: Laboratory diagnosis and treatment.

Granulomatous amebic encephalitis is a life-threatening central nervous system (CNS) infection caused by the free-living amoebae Acanthamoeba spp., Balamuthia mandrillaris and Sappinia pedata. The disease has a subacute to chronic onset affecting commonly the immunocompromised population with high mortality rate. The diagnosis of this disease entity requires high suspicion with appropriate sample collection and testing by the laboratory experts. Radiological investigations are nonspecific and commonly confused with CNS tuberculosis, neurocysticercosis, disseminated encephalomyelitis, viral encephalitis etc., delaying the accurate diagnosis of these cases. Early diagnosis plays a crucial role in the survival of these cases since appropriate management can be initiated. No single drug is effective; hence multiple antibiotics targeting various proteins or receptors are required for successful treatment. A combination of surgical and medical interventions involving multiple specialty experts is required to prevent death and morbidity in survivors.

Performance of polymerase chain reaction for the diagnosis of cystic echinococcosis using serum, urine, and cyst fluid samples.

INTRODUCTION: Cystic echinococcosis (CE) is a chronic zoonosis which presents with variable clinical manifestations. Currently the diagnosis of this disease is based on radiological findings and serological tests which lack specificity. Although antigen detection from the cyst fluid is the most specific, it is seldom done due to the complications involved. Detecting the presence of Echinococcus granulosus specific deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR) could provide a definitive diagnosis of CE. MATERIALS AND METHODS: An in-house PCR assay was devised to detect E. granulosus specific DNA in serum, urine and hydatid cyst fluid. The ability of the PCR to detect E. granulosus in the above mentioned samples were observed in comparison with other antigen and antibody detection tests. RESULTS: Serum samples from surgically confirmed patients of CE with ruptured cysts contained the corresponding DNA while the in the majority of cases who had an intact cyst had no DNA of E. granulosus in their serum. DNA of E. granulosus was not found to be excreted in urine. PCR performed equal to antigen detection ELISA while testing hydatid cyst fluid samples. CONCLUSIONS: Serum and urine might not serve as useful samples for the molecular diagnosis of cystic echinococcosis. However, PCR can be useful on serum samples to detect ruptured hydatid cysts and on hydatid cyst fluid to confirm the parasitic diagnosis.

Trichomoniasis: How do we diagnose in a resource poor setting?

BACKGROUND: Diagnosis of Trichomonas vaginalis vaginalis infection based solely on clinical symptoms and signs is unreliable because the spectrum of infection is broad and other sexually transmitted pathogens cause similar signs and symptoms. AIMS: Our study was undertaken to study the frequency of T. vaginalis infection in women presenting with vaginal discharge, to characterize the clinical features, and to study the sensitivity and specificity of microbiological investigations in the diagnosis of the same. MATERIALS AND METHODS: This was a hospital-based descriptive study done on 400 female patients with vaginal discharge attending the Gynecology out-patient department (OPD) of JIPMER, Puducherry, from May 2010 to July 2011. Women of age between 20 years and 50 years presenting with vaginal discharge irrespective of marital status, were included, and detailed history was elicited and thorough examination was performed. RESULTS: In 400 women presenting with vaginal discharge from Gynecology out-patient department (OPD) included in the study, T. vaginalis infection was found in 27 (6.75%) women. The risk factors for trichomoniasis included history of pre- or extramarital sexual contact in the woman or her partner, symptomatic partner, and alcohol consumption. A positive association with pelvic inflammatory disease was also observed. The most frequent symptoms included lower abdominal pain, dysuria, and dyspareunia. Combining of Whiff test, pH > 4.5, and pus cells in Gram-stained smear, the specificity in diagnosing the infection (97.3%) approached that of the reference standard, i.e., culture. On combining wet mount with Papanicolaou smear, the sensitivity increased to 92.6%, which was higher than that individually done. CONCLUSION: To conclude, diagnosis of T. vaginalis infection based solely on clinical symptoms and signs is unreliable, and combination of simple laboratory tests increases the diagnostic performance close to the reference standard (culture), especially in resource poor settings.

Blastocystis: Taxonomy, biology and virulence.

The unicellular protist Blastocystis has long been an unsolved puzzle for taxonomists, microbiologists and clinicians. Over the years, the organism has been bounced on and off the different branches of the tree of life due the possession of unique phenotypic characters intermediary to different organisms. The organism is polymorphic with only few of forms such as vacuolar, granular, amoeboid, and the cyst form being commonly known. However it could exist in other forms much more frequently than the widely known forms which could be missed by the unaware observer. Certain older concepts in the life cycle of Blastocystis although has been proven wrong are still being followed in various textbooks and other trustworthy internet sources. The causal role of Blastocystis in human disease has long been a subject of controversy. It is widely believed that certain subtypes of the organism are virulent. But this is not so as other factors are also involved in the clinical outcome of the infection. In these contexts, this review intends to shed light on the past misconceptions and the recent findings on the taxonomy, biology and the virulence of this organism.

Laboratory diagnosis of Taenia asiatica in humans and animals.

Taenia asiatica is a recently described species known to cause intestinal teniasis in humans and cysticercosis in animals. This species has close morphological resemblance to Taenia saginata and has a life cycle resembling Taenia solium, hence has been posing diagnostic dilemma and had been the reason for its comparatively late discovery. Recent diagnostic tools such as serological and molecular techniques have thrown light on its exact prevalence in the endemic countries. Hence introduction of utilization of these techniques in addition to the routine morphological analysis would be helpful in diagnosis of T. asiatica infections and early implementation of preventive measures.

Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis.

INTRODUCTION: Cystic echinococcosis (CE) is a zoonotic disease of humans with variable clinical manifestations. Imaging and immunological methods are currently the mainstay of diagnosis of this disease. Although the immunological tests for detection of anti-echinococcal antibodies have several disadvantages, they are widely being used. Antigen is far more superior than antibody detection test as they can provide a specific parasitic diagnosis. MATERIALS AND METHODS: A sandwich enzyme linked immunosorbent assay (ELISA) was designed using antibodies to 24 kDa urinary hydatid antigen for the detection of hydatid antigens in urine, serum and cyst fluid specimens. The performance of this novel test was compared with that of other hydatid antibody detection ELISA and enzyme immune transfer blot (EITB) using radiological and surgical confirmation as the gold standard. RESULTS: The antigen detection ELISA showed 100% sensitivity and specificity when tested with cyst fluid. On testing urine and serum, the antigen detection ELISA was found to be more specific than antibody detection ELISA. EITB was found to be the most sensitive and specific test. CONCLUSIONS: ELISA using polyclonal antibodies against 24 kDa urinary hydatid protein was moderately sensitive to detect hydatid antigen in serum and urine. Hence polyclonal antibodies to 24 kDa urinary hydatid antigen can be used as an alternative source of antibody to detect hydatid antigen in serum, urine and cyst fluid. In the present study, EITB was found to be highly specific test for detection of hydatid antibodiesin serum. 24 kDa protein was found to be specific and of diagnostic value in CE.

A review on diagnostic and preventive aspects of cystic echinococcosis and human cysticercosis.

Cystic echinococcosis and human cysticercosis have recently been included in the list of "neglected tropical diseases" by the World Health Organization (WHO). Both are zoonoses which are prevalent throughout the world and lead to considerable mortality, morbidity, and economic losses as well. This review deals with the disease burden of these two neglected cestode infections. Diagnostic modalities with their specific advantages and disadvantages have also been discussed. Recent developments in immunodiagnostic assays for the two diseases have been dealt with. Various control strategies including the use of veterinary vaccines have been highlighted.

Evaluation of Dot-ELISA and enzyme-linked immuno-electrotransfer blot assays for detection of a urinary hydatid antigen in the diagnosis of cystic echinococcosis.

BACKGROUND: Several serological assays are used for detection of a hydatid antigen in serum for diagnosis of cystic echinococcosis (CE). However, it requires technical expertise and is associated with the risk of acquiring blood-borne infections. Of late, interests have been shifted to other body fluids like urine, saliva, tear drops as alternate specimens in the diagnosis of CE. AIM: The aim of the study was to evaluate the dot enzyme-linked immunosorbent assay (Dot-ELISA) and electro-immunotransfer blot (EITB) for detection of a hydatid antigen in the urine for diagnosis of CE. MATERIALS AND METHODS: 100 ml of urine samples were collected from the patients with confirmed CE (n=30), patients with suspected CE (n=30), patients with other diseases (n=30) and healthy controls (n=30). A hydatid antigen in urine was detected by Dot-ELISA and EITB using only polyclonal antibodies raised against a complete homogenate hydatid (CHH) antigen in rabbits. RESULTS AND CONCLUSIONS: The Dot-ELISA using polyclonal antibodies showed a sensitivity of 53.33% and specificity of 96.66%, whereas EITB showed a sensitivity of 46.66%. The Dot-ELISA and EITB employing polyclonal antibodies showed no significant difference in sensitivity (P=0.426). Hence, the Dot-ELISA being a simple procedure can be used for detection of a hydatid antigen in urine for diagnosis of CE.

Antibiotic resistance pattern among common bacterial uropathogens with a special reference to ciprofloxacin resistant Escherichia coli.

BACKGROUND & OBJECTIVES: The resistance of bacteria causing urinary tract infection (UTI) to commonly prescribed antibiotics is increasing both in developing as well as in developed countries. Resistance has emerged even to more potent antimicrobial agents. The present study was undertaken to report the current antibiotic resistance pattern among common bacterial uropathogens isolated in a tertiary care hospital in south India, with a special reference to ciprofloxacin. METHODS: A total of 19,050 consecutive urine samples were cultured and pathogens isolated were identified by standard methods. Antibiotic susceptibility was done by Kirby Bauer disk diffusion method. The clinical and demographic profile of the patients was noted. RESULTS: Of the 19,050 samples, 62 per cent were sterile, 26.01 per cent showed significant growth, 2.3 per cent showed insignificant growth and 9.6 per cent were found contaminated. Significant association (P<0.001) of prior use of antibiotics in males, UTI in adults, gynaecological surgery in females, obstructive uropathy in males and complicated UTI in females with the occurrence of UTI with ciprofloxacin resistant Escherichia coli was noted. Significant association was noted in females with prior antibiotics, with prior urological surgery and in males with prior complicated UTI. There was no significant association with diabetes mellitus with the occurrence of UTI with ciprofloxacin resistant E. coli. Fluoroquinolone resistance was found to increase with age. INTERPRETATIONS & CONCLUSIONS: Ciprofloxacin resistance has emerged due to its frequent use. This resistance was seen more in the in-patients, elderly males and females. Also the resistance to other antibiotics was also high. Increasing antibiotic resistance trends indicate that it is imperative to rationalize the use of antimicrobials in the community and also use these conservatively.

Anti-Taenia solium larval stage Ig G antibodies in patients with epileptic seizures.

BACKGROUND: Cysticercosis is the most common differential diagnosis for epilepsy. The present study was carried out to assess the serological response among patients with epileptic seizures visiting JIPMER Hospital Puducherry. MATERIALS AND METHODS: A total of 934 serum samples were collected from patients with epileptic seizures. A standardized questionnaire was designed to obtain information on the demographic, socioeconomic, environmental, and behavioral characteristics related to the transmission of infection. An enzyme-linked immunosorbent assay (ELISA) was used to detect the anti-Taenia solium larval stage IgG antibodies. Samples found reactive and inconclusive by ELISA were further tested by the enzyme immunotransfer blot (EITB). RESULTS: The frequency of antibodies in the serum samples of the above-mentioned population was 16.2% by EITB. Anti-Taenia solium larval stage antibodies were detected in serum samples of 163 patients, out of which 27 (16.56%) patients belonged to the 0 - 15-year age group, 82 (50.30%) patients were in the 16 - 40-year age group, and 52 (31.90%) patients were above 41 years, respectively. Although the sera from males had higher OD values than those from females, the difference was not statistically significant. Out of 163 seropositive by ELISA, 152 (93.25%) were found to be positive by EITB. Out of the 152, 61 (40.13%) were farmers and 79 (51.97%) were office or factory workers. CONCLUSIONS: In conclusion, the results indicate a probable endemic situation and a high prevalence of cysticercosis in patients with epileptic seizures. Living in poor sanitary conditions seems to be an important factor related to human cysticercosis in Puducherry and the neighboring districts of Tamil Nadu.

Progress in the research on diagnosis and vaccines in amebiasis.

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.