# Estimating the Risk of Human Herpesvirus 6 and Cytomegalovirus Transmission to Ugandan Infants from Viral Shedding in Saliva by Household Contacts

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## Abstract

**:**

_{10}DNA copies/mL. The lack of association between oral CMV shedding and transmission is consistent with breastfeeding being the dominant route of infant infection for that virus. These affirm saliva as the route of HHV-6 transmission and provide benchmarks for developing strategies to reduce the risk of infection and its related morbidity.

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Study Cohort and Data

#### 2.2. Definitions of Exposure and Transmission Events

#### 2.3. Survival Analysis and Dose-Response Modeling

_{E}is the risk parameter associated with the exposure and the b0 parameter is a constant weekly risk parameter capturing the remaining risk not associated with exposure (i.e., when exposure was 0 DNA copies/mL). For the combined model, the weekly infection probability was calculated as follows:

_{0}. This was done using a likelihood ratio test, which is described in more detail in Appendix A. The predictions of the weekly risk were calculated from the individual (Equation (1)) and combined (Equation (2)) model using the fitted parameters using the range of exposures observed in the data.

_{50}) was calculated by solving for the exposure that gives a 50% infection risk given the fitted parameters using Equations (1) and (2). ID

_{25}and ID

_{75}were also calculated. For the combined model (Equation (2)), ID calculations were done for one exposure source at a time (the other was assumed to be 0 DNA copies/mL).

#### 2.4. Sensitivity Analysis

#### 2.5. Software, Data, and Code Availability

## 3. Results

#### 3.1. HHV-6 and CMV Transmission Occurred at High Rate Early During Infancy

#### 3.2. Secondary Children Shed at Higher Viral Loads than Mothers

_{10}DNA copies/mL) over the exposure period. For both viruses, secondary children uniformly had higher shedding frequencies and viral loads compared to mothers in the same household (Figure 2a–c). Summarized exposure measurements were not significantly correlated between mothers and secondary children within the same household, although there were modest positive correlations estimated for HHV-6 (Spearman correlation p-values > 0.05 for all, Table S2). Overall household exposure was assessed by taking the sum of maternal and secondary children viral loads at each week. In the majority of households, viral loads shed by secondary children contributed to the majority of the household exposure sum measure, comprising a median of 99.7% (IQR: 94.6–99.9%) of the CMV household sum and 91.2% (IQR: 80.5–98.8%) of the HHV-6 household sum (Figure S1).

#### 3.3. Correlation between household shedding and transmission to infants

#### 3.4. HHV-6, but not CMV, Acquisition in Infants was Associated with Viral Loads of Oral Shedding Exposures

#### 3.5. A Combined Exposure Model for HHV-6 Highlights Differential Risk between Mother and Secondary Children Exposures

_{50}), was 5.01 log

_{10}DNA copies/mL from maternal exposures compared to 5.92 log

_{10}DNA copies/mL from secondary children, which is almost a 10-fold difference. Using the single exposure models, ID

_{50}estimates showed a similar difference between sources: 4.92 and 5.87 log

_{10}DNA copies/mL from mother and secondary children, respectively. As exposure to CMV was not associated with infection risk, ID calculations were not performed for CMV.

#### 3.6. The Dose–Response Relationship between the Viral Load of HHV-6 oral Shedding Exposure and Weekly Infant Acquisition

_{10}DNA copies/mL for mothers or > 6 log

_{10}DNA copies/mL for secondary children) result in higher predicted risk. For CMV, as there was no risk attributable to exposure, the dose–response curves were flat, always predicting the 2% constant risk estimated from the model (Figure S2a).

_{10}DNA copies/mL range where the majority of transmissions were observed (Figure 3b). Of note, there were 107 exposure weeks with no measured household HHV-6 shedding measured and no instances of HHV-6 infections. In contrast, for CMV, there were 4 infections recorded among the 217 weeks with no household CMV shedding (Figure S2b). Using the model, the majority of combined exposures were associated with a low weekly probability of HHV-6 transmission (Figure 3c). The predicted risk increases dramatically for HHV-6 exposures higher than 5.5 log

_{10}DNA copies/mL from secondary infants and 4.5 log

_{10}DNA copies/mL from the mother. Though these high exposures were generally rare in the data, they were associated with an increased observed transmission risk, particularly when the mother had a detectable viral load. Together, the model and the data suggest that the majority of HHV-6 infections arise from repeated exposures to oral viral load shedding by all members in the household, but with varying degrees of contact efficiency. Episodes of high viral shedding by any household member substantially increases risk.

## 4. Discussion

_{50}) uncovered in this study, we established a baseline link between oral shedding and the weekly risk of transmission. Interventions that affect the susceptibility of infants (i.e., vaccination) would theoretically increase necessary quantities of exposure to result in infant infection, while antiviral therapy for infected contacts could lower exposure. The study of dose–response relationships before and after specific interventions could be used as a precise metric to develop and evaluate the effectiveness of interventions to prevent HHV-6 infection and its related morbidity.

## Supplementary Materials

## Author Contributions

## Funding

## Conflicts of Interest

## Appendix A

_{j}, is

_{j}denotes maternal exposure in the jth week), 3) secondary children exposure only (equation A2c; s

_{j}denotes secondary children exposure in the j

^{th}week), 4) total household exposure (sum in the j

^{th}week, m

_{j}+ s

_{j}) with common risk (equation A2d), and 5) full risk model with differential risk by mother or secondary children exposure (equation A2e).

_{k}(equation A2a – A2e), we assumed that at-risk infants were independent, and the risk associated with weekly exposure was unique from other exposures (i.e., non-infectious exposure weeks are exchangeable). That is, the accumulation of risk or survival probability does not depend on the order of exposures [46]. The overall negative log-likelihood function for all n infants was then defined as follows

^{th}infant was calculated across their unique exposure series (S(i,j)) for each j

^{th}week for all T

_{i}weeks without infection, and their infection week, T

_{i}+ 1, when infection was observed (${\Delta}_{i}$ = 1). From this formulation, the maximum likelihood estimates for the parameters were estimated by minimizing the negative log-likelihood.

## Appendix B

**Sensitivity analysis and final model estimates.**In our sensitivity analysis, the HHV-6 risk estimation for secondary children exposure was extremely sensitive to one household (Family AB, Figure A1a). Inclusion of that single household reduced the risk estimate by approximately 10-fold. Upon further inspection, this household had two secondary children, one of which was only present for half the study and had some of the highest exposure levels in the study (Figure A1b). Given the stability of estimation excluding this household (Figure A1a), they were removed from the final, presented model results. It is plausible that while that second secondary child represents high exposure risk, they never contacted the infant. We next evaluated sensitivity to interpolation. While interpolation rates ranged >50% for some households and was occasionally applied for more than 2 consecutive weeks, estimates were stable to inclusion of those households (Figure A2). No substantial differences were seen refitting the model using only infected households (Figure A2b). For CMV, all estimates across sensitivity analysis resulted in insignificant risk attributable to exposure (Figure A1 and Figure A2). For HHV-6, across all sensitivity analysis, risk estimates were different between mother and secondary children exposure.

**Figure A1.**Transmission model sensitivity analysis to individual households. (

**a**) Exposure risk coefficient estimate by exposure source and model re-estimated iteratively excluding one household at a time for both viruses; and HHV-6 was additionally evaluated completely excluding household AB. Estimates shown for both individual (single exposure) and combined exposure models. (

**b**) HHV-6 viral load (VL, DNA copies/mL) for household AB by exposure sources. Infant AB was infected following the final exposure.

**Figure A2.**Transmission model sensitivity analysis to exposure interpolation and infection status. (

**a**) Summary of interpolation by household (points) by maximum consecutive weeks interpolated and percentage of exposures weeks interpolation by exposure source for each virus. (

**b**) Estimated exposure risk coefficients for each virus allowing for a maximum total interpolation by household. For example, the risk estimate at 20% maximum interpolation includes only households with 20% interpolation or less. Estimates shown for both individual (single exposure) and combined exposure models. All households are included at 60%.

**Table A1.**Coefficient estimates and log likelihood from the survival analysis. For CMV, the log likelihood value was equivalent for all models (see equations (1 – 2) and Appendix A).

Virus | Model | Parameter | Estimate | -Log likelihood |
---|---|---|---|---|

HHV-6 | Constant risk (null) | b_{0} | 3.67 × 10^{−2} | 91.2 |

Secondary children | b_{0} | 2.10 × 10^{−2} | 84.8 | |

b_{S} | 9.13 × 10^{−7} | |||

Mother | b_{0} | 2.93 × 10^{−2} | 87.5 | |

b_{M} | 7.96 × 10^{−6} | |||

Household sum | b_{0} | 2.03 × 10^{−2} | 84.6 | |

b_{HH} | 9.12 × 10^{−7} | |||

Combined model | b_{0} | 1.82 × 10^{−2} | 84.2 | |

b_{M} | 6.56 × 10^{−6} | |||

b_{S} | 8.03 × 10^{−7} | |||

CMV | Constant risk (null) | b_{0} | 1.98 × 10^{−2} | 74.1 |

Secondary children | b_{0} | 1.94 × 10^{−2} | ||

b_{S} | 0 | |||

Mother | b0 | 1.94 × 10^{−2} | ||

b_{M} | 0 | |||

Household sum | b_{0} | 1.94 × 10^{−2} | ||

b_{HH} | 0 | |||

Combined model | b_{0} | 1.94 × 10^{−2} | ||

b_{M} | 0 | |||

b_{S} | 0 |

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**Figure 1.**Time taken for the transmission of human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV). Cumulative incidence curves for all transmission events to infants following birth stratified by maternal HIV status with censoring denoted by a plus. The P-values from log-rank tests comparing survival curves (one minus proportion infected) between infants with HIV+ and HIV- mothers are displayed in the bottom-right of each panel. The household exposure information was collected through one year (52 weeks; vertical, dashed black line). All events occurring after one year were right-censored for an estimation of transmission risk.

**Figure 2.**Comparisons of exposure patterns across households and among household members. Comparing viral exposure patterns for HHV-6 and CMV between mother and secondary children within a household by (

**a**) percent positive weekly swab measurements; (

**b**) mean log viral load (VL; DNA copies/mL); and (

**c**) maximum log viral load. The diagonal line in the scatterplot denotes equivalent summary measures between household mother and secondary children (i.e., the points below the line indicate lower measurements from the household mother). Shedding patterns by exposure source compared between households without and with transmission by (

**d**) the percent of positive weekly measurements; (

**e**) the mean log viral load; and (

**f**) the maximum log viral load. Boxes represent the interquartile range, stems represent values within 1.5 of the IQR, and points represent the raw data. All observed exposures measured in the infant’s first year of life and prior to transmission times are shown.

**Figure 3.**Estimation of HHV-6 acquisition risk from exposures by week using the combined exposure model (Table 2 and Table A1). (

**a**) Individual HHV-6 dose–response relationship for mother and secondary children oral viral load (VL, DNA copies/mL) exposures where bars depict the percentage of infections observed among total exposures in the data and lines depict estimated risk from the model. (

**b**) A heatmap depicting the distribution of total combined exposures in a given viral load bin from mothers and secondary children in a household. The text depicts the fraction of total infections over total exposures. (

**c**) A heatmap depicting the risk of HHV-6 infection from combined household mother and secondary children exposures estimated from the model. The text depicts the proportion of infections observed in the data using binned viral loads from (b) (equivalent to fraction displayed).

**Table 1.**Estimates of the weekly risk for each virus with no exposure (constant weekly risk) and by exposure source. Models of risk were fit without exposure (null model) and independently for each exposure source. Weekly risk with exposure includes constant risk from the model. The mean exposure was calculated among positive swabs. The P-values were calculated from the model to test if risk attributable to exposure is significantly different from null model without exposure risk (log-likelihood ratio test).

No Exposure | Mean Exposure | Maximum Exposure | |||||
---|---|---|---|---|---|---|---|

Virus | Exposure Source | Weekly Risk (%) | Log_{10} DNA copies/mL | Risk (%) | Log_{10} DNA Copies/mL | Risk (%) | p-Value |

HHV-6 | Null | 3.60 | |||||

Secondary children | 2.07 | 3.98 | 2.92 | 6.50 | 94.66 | <0.001 | |

Mother | 2.88 | 2.82 | 3.39 | 5.68 | 97.75 | 0.007 | |

Household sum | 2.01 | 3.94 | 2.79 | 6.50 | 94.63 | <0.001 | |

CMV | Null | 1.96 | |||||

Secondary children | 1.92 | 3.72 | 1.92 | 6.72 | 1.92 | 0.939 | |

Mother | 1.92 | 2.56 | 1.92 | 3.88 | 1.92 | 0.939 | |

Household sum | 1.92 | 3.77 | 1.92 | 6.72 | 1.92 | 0.939 |

**Table 2.**Estimates of weekly risk for each virus with no exposure (constant weekly risk) and by exposure source. Models of risk were fitted to adjust for both exposure sources and risks, which were calculated assuming that other exposure sources were 0 DNA copies/mL. The weekly risk with exposure includes constant risk from the model. P-values calculated from the model to test if risk attributable to exposure is significantly different from the null model without exposure risk (log-likelihood ratio test).

No Exposure | Secondary Children Exposure ^{1} | Maternal Exposure ^{1} | ||||
---|---|---|---|---|---|---|

Virus | Weekly Risk (%) | Mean Exposure Risk (%) | Maximum Exposure Risk (%) | Mean Exposure Risk (%) | Maximum Exposure Risk (%) | p-Value |

HHV-6 | 1.80 | 2.55 | 92.39 | 2.22 | 95.58 | <0.001 |

CMV | 1.92 | 1.92 | 1.92 | 1.92 | 1.92 | 0.997 |

^{1}Mean and maximum exposures (DNA copies/mL) shown in Table 1.

**Table 3.**HHV-6 weekly infectious exposure (ID, log

_{10}DNA copies/mL) associated with 25%, 50%, and 75% probability of infection (ID

_{25}, ID

_{50}, and ID

_{75}) estimated from the HHV-6 risk models by exposure source. For the exposure combined model, calculations assume a single exposure source. Exposure was not associated with CMV transmission and so IDs were not calculated.

HHV-6 Infectious Dose (ID, log_{10} DNA copies/mL) | ||||
---|---|---|---|---|

Model | Exposure Source | ID_{25} | ID_{50} | ID_{75} |

Individual | Secondary Children | 5.47 | 5.87 | 6.17 |

Mother | 4.51 | 4.92 | 5.23 | |

Combined | Secondary Children | 5.53 | 5.92 | 6.23 |

Mother | 4.61 | 5.01 | 5.32 |

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**MDPI and ACS Style**

Mayer, B.T.; Krantz, E.M.; Wald, A.; Corey, L.; Casper, C.; Gantt, S.; Schiffer, J.T.
Estimating the Risk of Human Herpesvirus 6 and Cytomegalovirus Transmission to Ugandan Infants from Viral Shedding in Saliva by Household Contacts. *Viruses* **2020**, *12*, 171.
https://doi.org/10.3390/v12020171

**AMA Style**

Mayer BT, Krantz EM, Wald A, Corey L, Casper C, Gantt S, Schiffer JT.
Estimating the Risk of Human Herpesvirus 6 and Cytomegalovirus Transmission to Ugandan Infants from Viral Shedding in Saliva by Household Contacts. *Viruses*. 2020; 12(2):171.
https://doi.org/10.3390/v12020171

**Chicago/Turabian Style**

Mayer, Bryan T., Elizabeth M. Krantz, Anna Wald, Lawrence Corey, Corey Casper, Soren Gantt, and Joshua T. Schiffer.
2020. "Estimating the Risk of Human Herpesvirus 6 and Cytomegalovirus Transmission to Ugandan Infants from Viral Shedding in Saliva by Household Contacts" *Viruses* 12, no. 2: 171.
https://doi.org/10.3390/v12020171