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BackgroundCryptosporidium sppare responsible for significant diarrheal morbidity and mortality in under-five children. There is no vaccine, thus a focus on prevention is paramount. Prior studies suggest that person-to-person spread may be an important pathway for transmission to young children. Here we describe a longitudinal cohort study of 100 families with infants to determine rates of cryptosporidiosis within households during the COVID-19 pandemic.MethodsFamilies living in Mirpur, Bangladesh with one infant age 6-8 months were enrolled and followed with weekly illness survey and stool testing for Cryptosporidium for 8 months.ResultsFrom December 2020 to August 2021, 100 families were enrolled. Forty-four percent of index children, and 35% of siblings had at least oneCryptosporidiuminfection. Shedding ofCryptosporidiumoccurred for a mean of 19 days (sd 8.3 days) in index infants, 16.1 days (sd 11.6) in children 1-5 years, and 16.2 days (sd 12.8) in adults. A longer duration ofCryptosporidiumshedding was associated with growth faltering in infants. There was a spike inCryptosporidiumcases in May 2021, which coincided with a spike in SARS-CoV-2 cases in the region.ConclusionIn this intensive, longitudinal study ofCryptosporidiuminfection in families we found high rates of cryptosporidiosis in infants and children, and prolonged parasite shedding, especially among malnourished children. These data support that transmission within the household is an important route of exposure for young infants, and that treatment of non-diarrheal infection to interrupt person-to-person transmission within the home may be essential for preventing cryptosporidiosis in infants.summaryCryptosporidiosis is a leading cause of morbidity and mortality among children. We followed 100 families with infants living in Bangladesh and studied the incidence ofCryptosporidiuminfection. We found prolongedCryptosporidiumshedding in stool was common among infants and adults.

Cryptosporidiosis is common in early childhood, and both diarrheal and subclinical infections are associated with adverse developmental outcomes. Improved therapeutic medications may help reduce the burden of cryptosporidial diarrhea; however, an effective vaccine would be better able to prevent the detrimental impact of both diarrheal and subclinical disease. A more complete understanding of naturally occurring immunity may further inform strategies to develop an effective vaccine. In this prospective cohort study of Bangladeshi children, greater fecal IgA at 12 months, but not plasma IgG, directed against two sporozoite-expressed, immunodominant and vaccine candidate antigens was associated with delayed time to subsequent cryptosporidiosis to 3 years of life. These findings extend prior work and further support the role of mucosal antibody responses in naturally developing protective immunity to Cryptosporidium.

Chronic exposure to infectious agents results in environmental enteric dysfunction—a significant contributor to childhood stunting. Low plasma tryptophan (TRP), increased kynurenine (KYN), and KYN–TRP (KT) ratio are associated with infections and chronic immune activation. We postulated that both these conditions are interlinked, and therefore aimed to identify the association between KT ratio and the linear growth of Bangladeshi children. A total of 480 stunted and at risk of being stunted children aged 12–18 months were enrolled and provided nutrition intervention for 90 days. Plasma samples were assessed using liquid chromatography tandem mass spectrometry to measure TRP and KYN concentrations. Multivariable linear regression with generalized estimating equations was applied to analyze association between the KT ratio and linear growth. Tryptophan, KYN, and KT ratio were significantly higher in stunted children than in children at risk of being stunted both at baseline and at the end of nutrition intervention. Following intervention, the median (interquartile range [IQR]) KYN concentration was significantly reduced from 4.6 (3.6, 5.4) µmol/L to 3.9 (0.3, 7.6) µmol/L, and median (IQR) KT ratio decreased from 104 (80.9, 131) to 92.8 (6.6, 247) in stunted children. We also found KT ratio to be negatively associated (coefficient = −0.7; 95% CI = −1.13, −0.26; P-value = 0.002) with linear growth. In addition, KYN and KT ratio were positively correlated with fecal neopterin and plasma C-reactive protein, whereas TRP was negatively correlated with both of these biomarkers and alpha-1-acid glycoprotein. Our findings imply that KT ratio is associated in the pathophysiology of stunting as well as with biomarkers of inflammation in Bangladeshi children.

Background Azithromycin is frequently used to treat shigellosis; however, clinical outcomes are uncertain. Methods We performed an observational cohort study in Bangladesh of patients with invasive diarrhea treated empirically with azithromycin. Susceptibility testing was performed by broth microdilution and disk diffusion post hoc on all Shigella isolates and clinical response was correlated with in vitro susceptibility. Results There were 149 Shigella culture-positive patients in the primary analysis. Infection with Shigella with decreased susceptibility to azithromycin was significantly associated with persistence of diarrhea at day 5 (31% vs 12%; relative risk [RR], 2.66; 95% confidence interval [CI], 1.34–5.28), culture positivity at day 5 or 6 (35% vs 5%; RR, 5.26; 95% CI, 1.84–14.85), and a higher rate of overnight hospitalization (58% vs 39%; RR, 1.49; 95% CI, 1.06–2.09). Shigella flexneri was more common than Shigella sonnei (58% vs 36%); however, S. sonnei constituted most of the isolates with decreased susceptibility to azithromycin (67%) and most of the multidrug-resistant strains (54%); thus, poor clinical outcomes were associated with S. sonnei. The current epidemiological cutoff for S. flexneri of ≥16 µg/mL to define decreased susceptibility to azithromycin was clinically predictive of poor outcome. Patients with S. sonnei and a low MIC (4 µg/mL) still had elevated rates of persistent diarrhea and culture positivity. Conclusions This study documents worse clinical outcomes for S. flexneri with decreased susceptibility to azithromycin, as well as S. sonnei, and supports the utility of susceptibility testing and clinical breakpoints for azithromycin. S. sonnei is an emerging drug-resistant threat. Clinical Trials Registration NCT03778125.

In this prospective cohort study of Bangladeshi children, greater fecal immunoglobulin A, but not plasma immunoglobulin G, directed against the Cryptosporidium sporozoite-expressed antigen Cp23 at 12 months of age was associated with delayed time to subsequent cryptosporidiosis. This finding suggests a protective role for mucosal antibody-mediated immunity in naturally exposed children.

We conducted a longitudinal study of cryptosporidiosis from birth to three years of age in an urban slum of Dhaka Bangladesh. Fecal DNA was extracted from monthly surveillance samples and diarrheal stool samples collected from 392 infants from birth to three years. A pan-Cryptosporidium qPCR assay was used to identify sub-clinical and symptomatic cryptosporidiosis. Anthropometric measurements were collected quarterly to assess child nutritional status. 31% (121/392) of children experienced a single and 57% (222/392) multiple infections with Cryptosporidium. Repeat infections had a lower burden of parasites in the stool (Cq slope = -1.85; p, Author summary Cryptosporidium is one of the top causes of diarrhea and growth faltering in Bangladesh infants. We discovered that a prior infection resulted in incomplete immunity that protected from diarrhea and growth faltering but not infection and was associated with mucosal IgA against a sporozoite surface protein Cp23. The most important implication of these findings is that a cryptosporidiosis vaccine may not need to achieve complete protection from infection to have a beneficial impact on child health.

Background The protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (ie, nondiarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection. Methods To characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples from 72 infants was evaluated using 16S ribosomal RNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype. Results Fecal microbiome composition was associated with diarrheal symptoms at 2 timepoints. Megasphaera was significantly less abundant in diarrheal samples compared with subclinical samples at the time of Cryptosporidium detection (log2 [fold change] = –4.3; P = 10–10) and prior to infection (log2 [fold change] = –2.0; P = 10–4); this assigned sequence variant was detected in 8 children who had diarrhea and 30 children without diarrhea. Random forest classification also identified Megasphaera abundance in the pre- and postexposure microbiota as predictive of a subclinical infection. Conclusions Microbiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection. Clinical Trials Registration. NCT02764918., Megasphaera abundance in the stool of Bangladeshi infants is associated with the development of diarrhea upon infection with the Cryptosporidium parasite.

Background Glucose hydrogen breath testing is a noninvasive test for small intestine bacterial overgrowth (SIBO). A positive glucose hydrogen breath test is common in children from low-income countries and has been found to be associated with malnutrition as measured by stunted growth. The microbiome associated with positive breath testing is relatively unstudied. Methods We performed 16 S V4 rDNA microbiome analysis on the stool of 90 Bangladeshi children aged 2 years from an impoverished neighborhood who were tested at the same time for SIBO by glucose hydrogen breath testing. Data were analyzed by linear discriminant analysis effect size with SIBO as the outcome. Any selected genera were tested individually by Wilcoxon’s rank-sum test to ensure that linear discriminant analysis effect size results were not outlier-skewed. Results Linear discriminant analysis effect size analysis identified Lactobacillus (linear discriminate analysis score, 4.59; P = .03) as over-represented in 15 out of the 90 children who were SIBO positive. Conclusions These results suggest that glucose hydrogen breath test positivity in children from low-income settings may be due to an upper intestinal Lactobacillus bloom, potentially explaining the association of SIBO with the gut damage and inflammation that leads to malnutrition.

Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards. DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction. For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.

BACKGROUND: Cryptosporidium is a major cause of childhood diarrhea. Current modes of cryptosporidiosis diagnosis involve procedures that are costly and require both a well-equipped laboratory and technical expertise. Therefore, a cost-effective, user-friendly, and rapid method for point-of-care detection of Cryptosporidium is desirable. METHODS: A total of 832 diarrheal stool specimens collected from 200 children aged

Oral vaccines for polio (OPV) and rotavirus are less effective in children in the developing world. The reasons for this are not well understood. We tested for risk factors for poor response to OPV in infants from an urban slum of Dhaka, Bangladesh. Diminished serum neutralizing response to OPV, but not failure of intramuscularly administered vaccines, was associated with malnutrition, diarrhea, and shorter breastfeeding duration. Children with malnutrition (WAZ

Enteric infections are associated with linear growth failure in children. To quantify the association between intestinal inflammation and linear growth failure three commercially available enzyme-linked immunosorbent assays (neopterin [NEO], alpha-anti-trypsin [AAT], and myeloperoxidase [MPO]) were performed in a structured sampling of asymptomatic stool from children under longitudinal surveillance for diarrheal illness in eight countries. Samples from 537 children contributed 1,169 AAT, 916 MPO, and 954 NEO test results that were significantly associated with linear growth. When combined to form a disease activity score, children with the highest score grew 1.08 cm less than children with the lowest score over the 6-month period following the tests after controlling for the incidence of diarrheal disease. This set of affordable non-invasive tests delineates those at risk of linear growth failure and may be used for the improved assessments of interventions to optimize growth during a critical period of early childhood.

We have developed recombinant fragment C based rapid point of care dipstick devices to assess tetanus immunization status using plasma or whole blood. The devices demonstrated specificity of 0.90 and sensitivity of 0.90 (whole blood) / 0.94 (plasma) at field sites in Bangladesh and The Gambia when compared to a commercial ELISA with the immune cut-off titer set as ≥ 0.1 IU/mL.

Entamoeba histolytica causes extensive mortality and morbidity worldwide through diarrheal disease and abscess formation in parenchymal tissues such as liver, lung, and brain. In contrast, other amoebae that infect humans include Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli, Entamoeba hartmanni, and Endolimax nana, which have been considered nonpathogenic commensals of the human gut [1–3]. Dientamoeba fragilis and Entamoeba polecki have been associated with diarrhea and Entamoeba gingivalis with periodontal disease [4, 5]. E. moshkovskii is genetically related to E. histolytica and E. dispar and is microscopically indistinguishable from them in its cyst and trophozoite forms [6]. This species of Entamoeba was first identified in sewage in Moscow by Tshalaria in 1941 [7] and was initially thought to be a free-living common protozoan species in anoxic sediments and in environments such as brackish coastal pools. The first human isolate was obtained from a resident of Laredo, Texas, who suffered from diarrhea, weight loss, and epigastric pain in 1961 [8]. This finding would seem to suggest and/or support that E. moshkovskii can be pathogenic. At first, this isolate was named E. histolytica Laredo strain and shared biological features with E. moshkovskii. Both the Laredo strain and E. moshkovskii grow at room temperature and were resistant to osmotic shock and to drugs used in the chemotherapy of amoebiasis such as emetine [9]. Subsequent molecular studies revealed that E. histolytica Laredo is identical with E. moshkovskii [10]. E. moshkovskii is a common Entamoeba infection in humans in some settings. It is composed of anywhere from as little as 1% to as high as 50% of the E. histolytica/E. dispar/E. moshkovskii complex parasites detected in fecal samples in limited studies from Australia, Bangladesh, India, Iran, Tanzania, and Turkey [6, 11–16]. These studies for the most part tested stool samples submitted to clinical microbiology laboratories from patients with gastrointestinal symptoms, suggesting that E. moshkovskii could cause disease. However, in HIV-1–infected individuals in northern Tanzania, E. moshkovskii was not associated with enteric symptoms nor immune status [17]. Thus, the ability of E. moshkovskii to cause disease in humans remains unclear. Here we tested the ability of E. moshkovskii to cause colitis and diarrhea in a murine model system, in which intracaecal inoculation with E. histolytica trophozoites into CBA/J, C3H/HeN, and C3H/HeJ mice leads to amebic colitis [18–20]. In addition, we tested in a longitudinal study of children in Bangladesh not only if E. moshkovskii was present in stool samples from infants with diarrhea, but whether the E. moshkovskii infection was newly acquired at the time of the diarrheal illness.

The contribution of interferon-gamma (IFN-gamma) to immunity from amebiasis was assessed in a three-year prospective study of children 2-5 years of age in an urban slum of Dhaka, Bangladesh. IFN-gamma produced by peripheral blood mononuclear cells stimulated with soluble amebic antigen was measured upon enrollment. Thirty-one of the 209 enrolled children had Entamoeba histolytica-associated diarrhea. Children who produced higher than the median level of IFN-gamma (median = 580 pg/mL) had longer survival without E. histolytica diarrhea/dysentery (log rank test P = 0.03) and a reduction in the risk of E. histolytica diarrhea/dysentery by more than half (Cox proportional hazard regression = 0.45, P = 0.04). When adjusted for stunting, the association between IFN-gamma and the time to the first episode of E. histolytica-associated diarrhea remained marginally significant (Cox proportional hazard regression = 0.49, P = 0.07). We conclude that production of IFN-gamma is linked to nutritional status and predicts future susceptibility to symptomatic amebiasis.

Children in developing countries are continually exposed to enteric pathogens [1–4]. This results in high rates of morbidity and mortality, especially in infants [5]. The outcome of an Entamoeba histolytica exposure can be diarrhea, dysentery, or amebic liver abscess, with the latter occurring mainly in men [6]. Disease, however, occurs in a minority of E. histolytica infections (approximately 20%) [6]. While both host and parasite factors contribute to outcome, they do not entirely explain susceptibility [7–9]. A prospective longitudinal study of enteric pathogens in a slum community in Dhaka, Bangladesh, has provided a rich data set to identify parameters important for protection and disease. It has been previously shown that children in this study cohort who are malnourished at birth are more susceptible to amebiasis and in general experience more-severe diarrhea [2]. There is evidence in support of both passive and active acquired immunity. Infants with mothers with high levels of breast milk anti-galactose/N-acetylgalactosamine (Gal/GalNAc) lectin immunoglobulin A (IgA) had fewer E. histolytica infections, and children with fecal IgA anti-Gal/GalNAc lectin IgA also had a lower incidence of new infection [10, 11]. Weaning and the introduction of supplementary food into the diet is known to trigger changes in the bacterial composition of the microbiome that include increases in Prevotellaceae and Bacteroides species [12, 13]. The first 2 years of life in general is a period of rapid immune and microbiome maturation, which, in this population, may be disrupted by malnutrition and environmental insult [13–15]. Here we describe the natural history of amebiasis in the first 2 years of life in infants from an urban slum in Dhaka. The cumulative incidence of amebic infection and diarrhea, the association of protection with anti-Gal/GalNAc lectin IgA, the impact of parasite burden on symptoms, and potential role of Prevotella copri in E. histolytica diarrhea are described.

Background Cryptosporidiosis is a common cause of infectious diarrhea in young children worldwide, and is a significant contributor to under-five mortality. Current treatment options are limited in young children. In this study, we describe the natural history of Cryptosporidium spp. infection in a birth cohort of children in Bangladesh and evaluate for association with malnutrition. Methodology/Principal Findings This is a longitudinal birth cohort study of 392 slum-dwelling Bangladeshi children followed over the first two years of life from 2008 to 2014. Children were monitored for diarrheal disease, and stool was tested for intestinal protozoa. Anthropometric measurements were taken at 3-month intervals. A subset of Cryptosporidium positive stools were genotyped for species and revealed that C. hominis was isolated from over 90% of samples. In the first two years of life, 77% of children experienced at least one infection with Cryptosporidium spp. Non-diarrheal infection (67%) was more common than diarrheal infection (6.3%) although 27% of children had both types of infection. Extreme poverty was associated with higher rates of infection (chi-square, 49.7% vs 33.3%, p = 0.006). Malnutrition was common in this cohort, 56% of children had stunted growth by age two. Children with Cryptosporidium spp. infection had a greater than 2-fold increased risk of severe stunting at age two compared to uninfected children (odds ratio 2.69, 95% CI 1.17, 6.15, p = 0.019) independent of sex, income, maternal body-mass index, maternal education and weight for age adjusted z (WAZ) score at birth. Conclusions/Significance Cryptosporidium infection is common (77%) in this cohort of slum-dwelling Bangladeshi children, and both non-diarrheal and diarrheal infections are significantly associated with a child’s growth at 2 years of age., Author Summary Diarrheal disease is a leading cause of death in young children worldwide. Cryptosporidium species are responsible for a large proportion of global burden of diarrhea. This study describes the natural history of cryptosporidiosis in a birth cohort of impoverished Bangladeshi children. Children were enrolled at birth and monitored for diarrhea twice a week for two years. Stool samples were tested for enteric protozoa. Children in this cohort had significant rates of malnutrition compared to the W.H.O. reference population, and extreme poverty was common. A majority of children were infected with Cryptosporidium spp, and we found that children who had at least one infection with Cryptosporidium spp during the two year follow up period were significantly more likely to have growth faltering by age 24 months. Cryptosporidiosis is a common infection in this cohort, and is associated with poverty and reduced growth during the first two years of life.

TO THE EDITOR: Diarrheal diseases have a major effect on global health, particularly the health of malnourished children (1). The enteric parasites Entamoeba histolytica and E. moshkovskii are potential causes of diarrheal disease in children (2). For the past 20 years, we have been studying Entamoeba infections in children from the urban slum of Mirpur in Dhaka, Bangladesh (3).

Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease.We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis.The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043).Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes.BillMelinda Gates Foundation Next Generation Molecular Diagnostics Project.

Giardia lamblia is the most prevalent human intestinal protozoan worldwide, but only a minority of infections result in diarrhea. We tested here whether the 2 major G. lamblia genotypes, assemblages A and B, differ in their propensity to cause disease. To determine whether an association exists between infection with assemblage A or B and diarrhea, 2534 Bangladeshi patients were enrolled in a case-control study. A total of 322 Giardia infections were identified and assayed for genotype by real-time polymerase chain reaction. Higher odds ratios for diarrhea were observed for assemblage A and A2 infections, whereas higher parasite DNA loads and a higher overall prevalence were observed for assemblage B infections. Our findings indicate that genotypic differences in virulence and fecundity may help to explain why not every Giardia infection results in disease, but they need to be confirmed in other urban populations of the developing world.

In this prospective cohort study, the presence of parasite-specific immunoglobulin A in breast milk was associated with protection of Bangladeshi infants from cryptosporidiosis and amebiasis. Our findings suggest that passive immunity could be harnessed for the prevention of Entamoeba histolytica and Cryptosporidium species infection in children living in endemic regions.

The study evaluated the usefulness of Leishmania-nested polymerase chain reaction (Ln-PCR) for diagnosis of kala-azar and assessed its role as a test of cure among kala-azar patients in Bangladesh. Peripheral blood buffy coat Ln-PCR was done in ninety-seven (97) clinically suspected patients of kala-azar, in forty (40) healthy controls from both endemic and non-endemic areas, and in forty-six (46) patients after completion of treatment with sodium stibogluconate (SSG). The Ln-PCR results were compared with Leishmania donovani parasite load graded by 1+ to 6+ in all smear-positive L. donovani cases. Out of 97 clinically suspected kala-azar patients, 94 were parasitologically confirmed. Ln-PCR was found positive in 91 of 94 parasitologically positive patients of kala-azar at diagnosis, indicating its diagnostic sensitivity as 97%. None of the controls was found positive for Ln-PCR, indicating its diagnostic specificity to be 100%. About 9% of kala-azar patients having been graded 1+ parasitic load had negative Ln-PCR results. After completion of treatment, Ln-PCR was positive in 4 patients (8.4%) out of 46 cases, indicating its role in demonstrating the absence of parasites 30 days after completion of treatment in 91.6% of the treated patients. This limited study suggests that Ln-PCR is a highly sensitive and specific test for the diagnosis of visceral leishmaniasis and can be used as a test of cure. Thus, efforts should be made to establish this useful method at least in the tertiary care hospitals and, if possible, at the district-level hospitals, especially in the endemic areas of Bangladesh.

The contribution of amebiasis to the burden of diarrheal disease in children and the degree to which immunity is acquired from natural infection were assessed in a 4-year prospective observational study of 289 preschool children in an urban slum in Dhaka, Bangladesh. Entamoeba histolytica infection was detected at least once in 80%, and repeat infection in 53%, of the children who completed 4 years of observation. Annually there were 0.09 episodes/child of E. histolytica- associated diarrhea and 0.03 episodes/child of E. histolytica- associated dysentery. Fecal immunoglobulin A (IgA) anti-parasite Gal/GalNAc lectin carbohydrate recognition domain (anti-CRD) was detected in 91% (183/202) of the children at least once and was associated with a lower incidence of infection and disease. We concluded that amebiasis was a substantial burden on the overall health of the cohort children. Protection from amebiasis was associated with a stool anti-CRD IgA response. The challenge of producing an effective vaccine will be to improve upon naturally acquired immunity, which does not provide absolute protection from reinfection.

Background The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools. Aims Three main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools. Methods Cysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins. Results A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets. Major Conclusions The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts., Author Summary We used tandem mass spectrometry to identify E. histolytica cyst proteins in 5 cyst positive stool samples. We report the identification of 417 non-redundant E. histolytica proteins including 195 proteins that were not identified in existing trophozoite derived proteome or EST datasets, consistent with cyst specificity. Because the cysts were derived directly from patient samples with incomplete purification, a limited number of proteins were identified (N = 417) that probably represent only a partial proteome. Nevertheless, the study succeeded in identifying proteins that are likely to be abundant in the cyst stage of the parasite. Several of these proteins may play roles in E. histolytica stage conversion or cyst function. Proteins identified in this study may be useful markers for diagnostic detection of E. histolytica cysts. Overall, the data generated in this study promises to aid the understanding of the cyst stage of the parasite which is vital for disease transmission and pathogenesis in E. histolytica.

The results of Entamoeba histolytica infections range from asymptomatic colonization to variable disease outcomes. However, markers that may predict infection outcomes are not known. Here, we investigated sequence types of a non-coding tRNA-linked locus R-R to identify surrogate markers that may show association with infection outcomes. Among 112 clinical samples-21 asymptomatic, 20 diarrhoea/dysentery and 71 liver abscesses-we identified 11 sequence types. Sequence type 5RR was mostly associated with asymptomatic samples, and sequence type 10RR was predominantly associated with the symptomatic (diarrhoea/dysentery and liver abscess) samples. This is the first report that identifies markers that may predict disease outcomes in E. histolytica infection.