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<0.0001) and were more likely to be sub-clinical (Chi square test for trend; p = 0.01). Repeat infections were associated with the development of growth faltering (Pearson correlation = -0.18; p = 0.0004). High levels of fecal IgA antibodies against the Cryptosporidium Cp23 sporozoite protein at one year of life were associated with a delay in reinfection and amelioration of growth faltering through three years of life (HAZ IgA high responders -1.323 ± 0.932 versus HAZ -1.731 ± 0.984 p = 0.0001). We concluded that nonsterile immunity to cryptosporidiosis in young children was associated with high levels of mucosal IgA anti-Cp23 and protection from diarrhea and growth faltering. Trial Registration: NCT02764918.

There is no vaccine to protect from cryptosporidiosis, a leading cause of diarrhea in infants in low- and middle-income countries. Here, we comprehensively identified parasite antigens associated with protection from reinfection. A Cryptosporidium protein microarray was constructed by in vitro transcription and translation of 1,761 C. parvum, C. hominis, or C. meleagridis antigens, including proteins with a signal peptide and/or a transmembrane domain. Plasma IgG and/or IgA from Bangladeshi children longitudinally followed for cryptosporidiosis from birth to 3 years of age allowed for identification of 233 seroreactive proteins. Seven of these were associated with protection from reinfection. These included Cp23, Cp17, Gp900, and 4 additional antigens — CpSMP1, CpMuc8, CpCorA and CpCCDC1. Infection in the first year of life, however, often resulted in no detectable antigen-specific antibody response, and antibody responses, when detected, were specific to the infecting parasite genotype and decayed in the months after infection. In conclusion, humoral immune responses against specific parasite antigens were associated with acquired immunity. While antibody decay over time and parasite genotype-specificity may limit natural immunity, this work serves as a foundation for antigen selection for vaccine design.

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.

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.

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.

Infections and persistent immunological activation are linked to increased kynurenine (KYN) and the KYN-to-Tryptophan (TRP) or KT ratio and may be critical factors in undernutrition. We sought to determine the association between the KT ratio and adult malnutrition, as well as investigate if nutritional supplementation had any influence on the decrease of the KT ratio. A total of 525 undernourished adults aged 18–45 years were recruited and provided a nutrition intervention for 60 feeding days. TRP and KYN concentrations were determined from plasma samples using LC-MS/MS. At baseline, the median (interquartile range (IQR)) TRP, KYN and KT ratios were 24.1 (17.6, 34.3) µmol/L, 0.76 (0.53, 1.18) µmol/L and 30.9 (24.5, 41.7), respectively. Following intervention, the median (IQR) KYN and KT ratios were significantly reduced to 0.713 (0.46, 1.12) µmol/L and 27.5 (21.3, 35.8). The KT ratio was found to be inversely linked with adult BMI (coefficient: −0.09; 95% CI: −0.18, 0.004; p-value = 0.06) but not statistically significant. Additionally, Plasma CRP was correlated positively, while LRP1 was inversely correlated with the KT ratio. Our data suggest that in Bangladeshi adults, the KT ratio is not related to the pathophysiology of malnutrition but correlated with inflammatory and anti-inflammatory biomarkers, and the ratio can be reduced by a nutrition intervention.