Impact of sampling depth on pathogen detection in pit latrines

Wastewater based epidemiology (WBE) is increasingly used to provide decision makers with actionable data about community health. WBE efforts to date have primarily focused on sewer-transported wastewater in high-income countries, but at least 1.8 billion people in low- and middle-income countries (LMIC) use onsite sanitation systems such as pit latrines and septic tanks. Like wastewater, fecal sludges from such systems offer similar advantages in community pathogen monitoring and other epidemiological applications. To evaluate the distribution of enteric pathogens inside pit latrines–which could inform sampling methods for WBE in LMIC settings unserved by sewers–we collected fecal sludges from the surface, mid-point, and maximum-depth of 33 pit latrines in urban and peri-urban Malawi and analyzed the 99 samples for 20 common enteric pathogens via multiplex quantitative reverse transcription PCR. Using logistic regression adjusted for household population, latrine sharing, the presence of a concrete floor or slab, water source, and anal cleansing materials, we found no significant difference in the odds of detecting the 20 pathogens from the mid-point (adjusted odds ratio, aOR = 1.1; 95% confidence interval = 0.73, 1.6) and surface samples (aOR = 0.80, 95% CI = 0.54, 1.2) compared with those samples taken from the maximum depth. Our results suggest that, for the purposes of routine pathogen monitoring, pit latrine sampling depth does not strongly influence the odds of detecting enteric pathogens by molecular methods. A single sample from the pit latrines’ surface, or a composite of surface samples, may be preferred as the most recent material contributed to the pit and may be easiest to collect.


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We purposively enrolled participants in urban Blantyre and peri-urban Mzuzu, Malawi  Figure S1). If there was visible solid waste in the pit that was likely to clog the Gulper, the 123 waste was carefully removed using a metal pitchfork while attempting to minimize any mixing 124 of the pit contents ( Figure S2). Next, a Gulper was lowered into the pit until the full length of the 125 Gulper (approximately 1.5m) was submerged ( Figure S3). The first "pump" of sludge was 126 assumed to have been the sludge that filled the Gulper as it was lowered and was not saved.

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Then, the second "pump" of sludge was collected in a bucket and considered as the sludge from 128 the bottom of the pit. When approximately half of the pit was emptied, another "pump" of sludge Malawi (Blantyre samples) ( Figure S4).

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On the same day as sample collection from the household's latrine, we interviewed an 135 adult member of the household regarding the characteristics and typical use of their pit latrine.

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Direct observation of the characteristics and condition of the latrine was guided by a checklist.  similarity coefficient is 67% (Equation 1). Further, we calculated 95% confidence intervals 163 around the Jaccard similarity coefficient by bootstrapping our data with 10,000 iterations.

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Comparing the three sampling depths, the Jaccard similarity coefficient indicated that the  (Table S3).

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Adjusted for household population, latrine sharing, the presence of a concrete floor or 220 slab, water source, and anal cleansing materials, there was no significant effect of depth on the  households who reported a public tap as their water source; using water for anal cleansing was 232 not associated with pathogen detection compared to using toilet paper (aOR = 1.8, 95% CI: 0.79, 233 4.2); and latrines with concrete floors or slabs were not associated with pathogen detection (aOR 234 = 0.89, 95% CI: 0.42, 1.9) compared to latrines without concrete floors or slabs (Table 3).
235 We observed a high degree of homogeneity in enteric pathogen detection between fecal 239 sludge samples collected at the top, middle, and bottom of pit latrines in urban and peri-urban

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We observed that increasing household population was associated with an increased odds of 273 pathogen detection. This finding was expected as the more people contribute waste to a pit 274 latrine the greater the probability that at least one person from the household would shed a 275 specific pathogen into the pit. In contrast, a study in low-income urban Maputo, Mozambique did 276 not find an association between pathogen detection and an increasing population within housing 277 compound (23). In addition, we found that having a water source inside the compound or home 278 reduced the odds of pathogen detection compared to relying on water from a public tap. Having a 279 water source inside the home may result in increased water use for personal hygiene or 280 consumption, which a 2015 systematic review found was generally associated with reduced 281 gastrointestinal infection and diarrheal disease (51).

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There are several important limitations to this study. First, by removing the solid waste from 283 some pits we may have inadvertently mixed the fecal sludge and we did not sterilize the Gulper 284 between collection of the bottom and middle samples. Mixing before sample collection and cross 285 contamination during sampling may have occurred and might explain why we observed that 286 these two samples were more closely aligned with each other than with the top samples.

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However, solid waste removal was necessary to use the Gulper, sterilization of the Gulper was 288 not logistically practical, and the mass of fecal sludge flowing through the Gulper was 289 substantially greater than the mass of fecal sludge that may have stuck to the walls of the Gulper 290 tube. Next, we used a quantal assay that determined the presence or absence of each pathogen.

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There may be important differences in pathogen concentrations between the depths assessed, 292 which could influence sampling methods for waste-based epidemiology efforts from pit latrines.

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Although we frequently co-detected pathogens, we cannot definitively explain the causal 294 mechanism behind our findings. A next step would be to conduct tracer studies in latrines and 295 septic tanks in LMICs to assess mixing inside these systems. Our sample processing methods 296 used centrifugation and discarded the resulting supernatant. While we frequently detected all 297 viral pathogens, it is plausible that these methods underestimated viral pathogen prevalence by 298 not analyzing the supernatant as well as the solids. Further, numerous studies have characterized 299 pathogen decay in wastewater, including the decay of nucleic acids which are detected via PCR-300 based methods like those used in this study. Similar decay experiments are needed for fecal 301 sludges. As well, we did not include household wealth or income as a confounder in our 302 regression analysis because such an assessment would have added to the length and complexity 303 of the questionnaire. It is likely some of the significant associations we found, such as the 304 association between pathogen detection and water source, were confounded by socioeconomic 305 status. Finally, as a cross sectional study, we were unable to assess pathogen signals over time, 306 which are necessary for interpretation and use in surveillance efforts.

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Though we most frequently co-detected pathogens in the matched samples from the middle 308 and bottom of pits, sludge samples from the top of pit latrines may be desired for surveillance 309 efforts. Operating a Gulper is messy and requires specialized training. Other sampling methods 310 exist to collect sludge from the middle or bottom of pit latrines (23,33), but still require 311 specialized equipment (e.g. a Wheaton sub-surface sampler). The simplicity of using a hand  The authors declare that they have no known competing financial interests or personal 325 relationships that could have appeared to influence the work reported in this paper. 1. Figure S1. Hand scoop for fecal sludge collection 328 2. Figure S2. Solid waste removal from a pit latrine 329 3. Figure S3. Operating the Gulper 330 4. Figure S4. Aliquoting a fecal sludge sample 331 5. Text S1. Methodology for total nucleic acid extraction from fecal sludges.