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* Mel and Enid Zuckerman Arizona College of Public Health, University of Arizona, Tucson AZ 85724;
Department of Pediatrics, University of Arizona, Tucson, AZ 85724;
** Tribhuwan University, Kathmandu, Nepal;
National Eye Hospital, Kathmandu, Nepal;
U.S. Centers for Disease Control and Prevention, Atlanta, GA 30341;
Optical Sciences Center, University of Arizona, Tucson, AZ 85721; and
# Department of Biochemistry, University of Arizona, Tucson, AZ 85724
3To whom correspondence should be addressed. E-mail: taren{at}email.arizona.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • xerophthalmia • vitamin A • pregnancy • night blindness • epidemiology
Night blindness is considered one of the earliest and milder symptoms of vitamin A deficiency (1,2). Night blindness is most prevalent in young children and in pregnant women during their second and third trimesters. Oral responses to questions about being able to see during dusk or at night have been used to determine the night blindness status of pregnant women, with prevalence rates in Asia being between 10 and 50% (3,4). A quantitative noninvasive method to determine the functional outcome of vitamin A deficiency (VAD)4 will assist with large-scale needs assessments and evaluations of vitamin A distribution programs. Several methods that use an objective measure of night blindness have now been developed and tested (5–10). Each of these tests was shown to be useful under specific conditions, but all have some shortcomings.
Our research team has been working on the development of the Night Vision Threshold Test (NVTT) that uses a low-cost and very portable instrument to measure night blindness (11,12). In our initial report of the NVTT with children at the Mwanamugimu Nutrition Unit at Makerere Medical School in Kampala, Uganda, the NVTT was correlated (r = 0.41, P < 0.05) with serum vitamin A (SVA) for children whose concentrations were <0.35 µmol/L. In that study, a dial was used to change the illuminance emitted from the NVTT instrument (11). In a follow-up study in Sanku, Nepal, preschool children were tested with an NVTT instrument that was modified to have 5 preset settings. In that study, 93% of children who initially failed the NVTT improved their NVTT score after receiving a vitamin A supplement of 209 µmol (12).
We report the results of a study conducted with pregnant women in Kathmandu, Nepal. The purpose of this study was to determine how the NVTT and reported night blindness compared with SVA in pregnant women. Furthermore, we wanted to determine whether it would be possible to develop an algorithm that could be used to estimate the prevalence of low SVA concentrations for a community.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The Nepalese obstetrical nurses informed women during their prenatal clinics that there was a study being conducted and that they could participate if they wanted to. If there was an affirmative response the research nurses recruited them to participate in the study. The study was explained to the women and written subject consent was obtained. Women were then interviewed to obtain demographic and health data. Three women refused to consent to participate in the study; 1 woman did not participate because her husband was present and did not want her to participate and 2 women said they would be afraid of the dark. Three additional women refused due to time constraints. Three women dropped out of the study after consent was obtained. Four women refused to have their blood drawn. A total of 1401 women participated in the NVTT and reported their night blindness status.
Maternity nurses, who were members of the research team, conducted all the interviews with the women in Nepali. A brief demographic and obstetrical history interview was conducted, including current status of night blindness. Following the interview, the NVTT was conducted. If a woman failed the NVTT (case), a blood sample was obtained. A blood sample for vitamin A analysis was also collected from the next woman who could be age- and gestation period–matched and who passed the NVTT (control). If more than 1 woman was present who could be matched with a case at the same time and 1 had reported night blindness, the case was the woman who reported night blindness. This resulted in recruiting 21 controls of 173 who reported night blindness but passed the NVTT.
Women were excluded from participation who were <24 wk gestation, who were unable to cooperate due to fear of the dark, who had difficulty concentrating on the test procedure, or who were blind or could not respond to verbal questioning. All other women were eligible for the study. An ophthalmic examination was conducted to look for signs of xerophthalmia and referral to an ophthalmologist was made for any woman identified as needing medical follow up. However, none of the women had clinical signs of xerophthalmia. Nutrition education on vitamin A was provided to women following their participation in the study. Approval for the study was obtained from the Institutional Review Board Human Subjects Committee at the University of Arizona, from the Nepal Health Research Council in Kathmandu, Nepal, and from the Hospital Director at the Prashuti Griha Royal Maternity Hospital.
Reported night blindness. There is no single method for determining night blindness. We used a single question: "Do you have problems seeing in the dark?" This is the standard question used at the National Eye Hospital and is known to be understood by patients. We decided to use this single question after several consultations within the investigative team that included a female Nepalese ophthalmologist (N.S.) and the obstetrical nursing staff. This single question was used to minimize any leeway for the interviewers that may lead to variations in probing about night blindness. Furthermore, a single local term was not used, given the metropolitan setting, because women who attended the hospital were not from a single ethnic group. However, on the few occasions that this question was not understood, the research nurse explained that she wanted to know whether the woman was now having trouble seeing in the dark compared with before she became pregnant.
The NVTT. Following the interview, the women were dark-adapted for 10 min in a totally darkened room and the NVTT was administered. The members of the research team who conducted the NVTT were unaware of how the women responded to questions regarding night blindness.
The NVTT instrument is a small, hand-held, portable, battery-powered instrument. It measures 14 x 11 x 3.5 cm in size and weighs 450 g (Fig. 1). It projects light that is 30 cm in diameter at 3.3 m by changing the electrical current provided to a light-emitting diode (LED) through a series of switches. In order to provide consistent illumination, each switch selects a different resistor in the current-regulated power supply of the NVTT instrument. The NVTT instrument requires a minimum of 6 V for correct operation and is set up with six 1.5-V AA batteries (9 V). A low-battery indicator light is activated when the battery power decreases to <7 V. The indicator light was not activated after >1000 tests. Seven switches on the exterior of the instrument project a light that decreases linearly in degrees of illuminance on a log scale (Table 1). An 8th switch provides a training setting. The LED for the NVTT emits a light with a wavelength of 0.50–0.57 µm in the green spectrum. This wavelength was chosen because the scotopic response of the eye (night vision) peaks at 0.51 µm. The photopic response of the eye (day vision) peaks at 0.55 µm. The incident light is measured in Lux (1 Lux is 1 lumen per square meter). The illuminance of the NVTT ranges from 475 mLux at the brightest test setting to a minimum of 0.3 mLux for the dimmest setting. The switches are numbered from 1 to 7 with lower numbered switches giving brighter light. Each person is given a score based on the dimmest light that they can see.
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The NVTT is administered by having a subject sit in a completely blackened interior room with the door cracks sealed for 10 min so that they can become dark-adapted. After dark adaptation, the subject is shown the test light to get her attention and show her what to recognize when the light is shown. Subjects must respond not only that they can see the light, but also where is the light projected on the screen (left, center, right). The placement of the light is random so subjects are not able to identify a pattern to where the light is projected. Following this introduction, the dimmest beam is projected. If the person is able to see the dimmest light the test is completed. If the person is not able to see the dimmest light, the next brightest light is displayed. If the person is able to see this light, then the dimmer light is shown a second time. If the subject reports again she cannot see the light, the brighter light is shown again. This sequence is repeated with each increasing light until the tester identifies the dimmest light that the subject can see on 2 different occasions.
The NVTT can easily be administered to 12 subjects/h when subjects are dark adapted in groups of 4 subjects. This allows for screening large populations as part of a surveillance system. After dark-adaptation, the NVTT test takes less than 1 min to perform.
Prior to the start of the study, training sessions were conducted to standardize the protocol that would be used to administer the NVTT. Three Nepalese obstetric nurses were trained to administer the NVTT by 1 of the investigators (DWG) who remained onsite throughout the data collection period. They first practiced giving the NVTT with each other using the standard procedure. This allowed the testers to become familiar with the instrument, the location of switches, directing the light toward a screen, giving instructions to subjects, and recording the results. This was followed by a reliability study to determine whether the scores would be consistent within and between observers. The initial within and between measures were conducted with the first 10 women who were recruited into the study, of whom 4 had failed the NVTT. In each of the 10 cases the values were the same between observers after 10 and 15 min of dark adaptation. During the study, the next 5 women who failed the test were also retested by the second observer to verify the score. These also remained the same, suggesting that the reliability of the NVTT is acceptable and that there was no learning effect on the results by taking the NVTT several times within a short period of time. Quality control and assurance continued throughout the study with discussions that reviewed the procedures with the onsite investigator and random retesting of women who had failed the NVTT.
Serum vitamin A concentration. Blood samples (4 mL) were drawn using standard venipuncture procedures on the day subjects completed the NVTT. The samples were immediately placed in a customized light-proof box for the vacutainers until the blood coagulated. Within 2 h of collection, the blood samples were centrifuged to obtain serum. Serum was then partitioned into 3-mL screw-cap vials and placed in a Dewar flask under nitrogen at –70°C. Samples were transported to a deep freeze (–20°C) until transported to the United States. Samples were hand carried in a container with dry ice to the Arizona Health Science Center where they were cataloged and prepared for shipment to the U.S. Centers for Disease Control and Prevention nutrition laboratory in Atlanta, Georgia, for final analysis. Samples shipped frozen to the nutrition laboratory for analysis arrived frozen. All samples were analyzed within 12 mo of collection. SVA concentrations were measured using HPLC with photodiode array detection using a modification of the method described by Sowell and colleagues (13).
Blood samples were obtained from a total of 217 cases that failed the NVTT and 216 matched controls. However, only a total of 350 blood samples were analyzed for this study (177 cases and 173 controls). Eighty-three samples had to be discarded due to a power outage and resulting thawing that occurred while in storage in Nepal or hemolysis that occurred with a few blood draws. An analysis comparing the demographic characteristics of subjects whose blood samples were not used in this analysis with those that were used in the analysis indicated that there were no significant differences in regard to age, gestation, or parity within each group (passed NVTT or failed NVTT) or between the 2 groups. Approximately the same number of cases and control blood samples were discarded.
Data analysis. A sample size of 200 per group was targeted to conduct a paired Student’s t-test to determine differences (P < 0.05) between cases and controls with a power of 80%. Chi square tests were conducted to compare proportions between groups. Correlations and simple regression analyses were performed to determine the association between the NVTT results and SVA concentrations. Receiver operating characteristic (ROC) analysis and classic sensitivity and specificity analyses were conducted using the STATA ROCTAB to determine how the NVTT served as a test for identifying low vitamin A concentrations. Sensitivity and specificity values were calculated in regard to the relative prediction of having an SVA concentration of <0.35 and <0.70 µmol/L. Values in the text are means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The SVA concentration did not differ between the women who reported night blindness and those who did not (Table 4). SVA concentrations < 0.35 µmol/L were found in 2.1% of the women who reported night blindness and in 1.0% in the women who did not (P > 0.05). Similarly, the percentage of women who had an SVA < 0.70 µmol/L did not differ between those who reported night blindness (10.6%) and those who did not (7.3%).
The ROC analyses were conducted with regard to SVA concentrations using 2 different cut-points: SVA < 0.35 µmol/L and SVA < 0.70 µmol/L. Failure to pass the NVTT was the criterion for classifying vitamin A deficiency. This analysis indicated that the NVTT had a sensitivity of 0.74 and specificity of 0.51 for predicting an SVA < 0.70 µmol/L and a sensitivity of 100.0 and specificity of 0.50 for predicting an SVA < 0.35 µmol/L. Reported night blindness had a sensitivity 0.18 of and a specificity of 0.87 for predicting an SVA < 0.70 µmol/L and a sensitivity of 0.25 and specificity 0.87 for predicting an SVA < 0.35 µmol/L.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The SVA concentrations for the women in this study were slightly greater than those reported for women in rural Nepal (4,14,15). Christian et al. (16) indicated that the SVA concentrations of rural Nepalese pregnant women who reported night blindness had an SVA concentration of 0.72 µmol/L compared with 1.03 µmol/L for women who did not report night blindness. Our values are closer to 1.20 µmol/L in this urban population and cases of night blindness were also present. Christian et al. determined that 51.8% of women who reported night blindness had an SVA < 0.70 µmol/L compared with 21.0% of non-night-blind women. In our study the percentage of women who had SVA concentrations < 0.70 µmol/L did not differ between women who reported night blindness and those who did not. Brown and colleagues also recently reported that the SVA concentrations did not differ between women who responded negatively or positively to questions about having night blindness (14).
One possible reason for fewer women reporting night blindness in our study compared with the percentage who failed the NVTT could be that the study was conducted in an urban environment where it may be more difficult to recognize night blindness. Urban areas have more lighted areas and fewer completely dark areas than rural areas, perhaps making complete dark adaptation not as necessary. Thus, some women might not have recognized that they had a mild form of night blindness compared with women who live in rural areas where electricity and other sources of light are much more limited or nonexistent.
Results from national Nepalese studies on the prevalence rate of night blindness during pregnancy have varied from 6 to 18% (17,18). The current study suggests that a prevalence rate of 16% could be present among poor pregnant women in Kathmandu who received prenatal care from the maternity hospital. Within the national vitamin A surveillance program, district data indicated that although the Kathmandu district had high rates of women with low SVA concentrations, the prevalence of night blindness was lower than in other parts of the country (18). These data also suggested that underreporting of night blindness may occur in more urban areas.
Correlations between the NVTT and SVA were greater than in previous reports using other objective measures of night blindness. Brown et al. (14) reported a correlation coefficient of –0.13 between the pupillary response test and SVA concentrations. Congdon et al. (10) reported a significant regression coefficient between the pupillary response test and SVA concentration in women who had SVA concentrations similar to those found in our study, but a graph of the relationship between the SVA and the dark adaptation threshold does not suggest a much better correlation coefficient than that reported by Brown and colleagues (14). Of note, the regression coefficient reported was not significant for women who received prenatal doses of vitamin A and had a greater mean SVA concentration (10). The NVTT also appears to be a better test for classifying pregnant women with very low SVA concentrations compared with self-reported night blindness. This was similar to a study that used the NVTT to test children, in which the correlation between the NVTT score and the SVA concentration was only significant for children with an SVA concentration < 0.35 µmol/L (11).
A history of night blindness remains the most frequently used indicator for the earliest and mildest expression of clinical xerophthalmia (1,19). The history of night blindness, however, is very subjective. It does not determine the degree or severity of impaired dark adaptation and its accuracy is highly questionable in infants or very young children. In some cultures where night blindness is more prevalent, special words have been developed to describe it. We suggest that there are gradations as to how well one can see in the "dark" and that when a person recognizes that he or she is having problems is dependent on his or her perception. Therefore, oral self-reports about night blindness may be unreliable and cause policy makers to make programmatic decisions based on erroneous data.
In the current study, the aggregate inability to not see successive levels of brightness was associated with decreased vitamin A status. Of the women who were categorized as having night blindness with the NVTT, 20% were not able to see a light brighter than the dimmest light. This indicated that the night blindness was worse for some women compared with other women. Thus, the degree of night blindness varied among women and the poorer the night vision, the lower the SVA concentration. This relationship was also reported using the pupillary response test (10). Although the sensitivity and specificity associated with failing the NVTT, e.g., not seeing the dimmest light, were moderate when the NVTT used several cut-points to estimate low SVA concentrations it will be able to provide a better estimate of the prevalence of VAD than reported night blindness that is dichotomous (i.e., present or not present). Given these results, we conclude that the algorithm presented in Table 5 could be used to predict the population of women with low SVA concentrations. However, this algorithm and the regression equation developed from this study to estimate the percentage of women with low SVA concentrations using the NVTT should be verified with additional studies and would be best done in each population that uses the NVTT. We propose that a probability approach is a better method for estimating the prevalence of vitamin A deficiency in a population compared with the dichotomous approach of classifying women as having or not having night blindness (20). The low specificity from our findings was primarily due to scores associated with brighter levels of light. It would be possible to increase the specificity by only identifying subjects as having night blindness when they could not see the dimmer levels of light (scores 1–4). However, with regard to programmatic decisions, this would mean that more women with VAD would not get low doses of vitamin A. The final decision about who would receive vitamin A doses based on an NVTT score will need to be made reagarding the overall prevalence of VAD and the financial resources available to a program.
Recommendations for programs to improve the vitamin A status of pregnant and postpartum women living in vitamin A–deficient areas include providing supplements of 10.4 µmol retinol equivalents on a daily basis or a supplement of 26.1 µmol retinol equivalents weekly (21). Postpartum administration of vitamin A includes providing mothers with 418 µmol of retinol equivalent as 2 doses of 209 µmol at least 1 d apart as soon after delivery as possible (19). The NVTT is an objective, low-cost, and portable method that can be used to conduct baseline studies and evaluate the outcome of these recommendations in various parts of the world. Our reliability studies also indicate that any learning effect that occurs with multiple administrations of the NVTT does not change the NVTT scores.
The current study had some shortcomings. There is a need for further testing of the NVTT under various other conditions to fully assess its utility. We did not measure disease state or inflammation in this study. However, those measurements would only increase the strength of the relation between the NVTT and vitamin A status by removing additional known causes of variance. Future studies with the NVTT should include its responsiveness to vitamin A supplements during pregnancy and studies to validate the probability approach should be undertaken to obtain the most appropriate weights for predicting the proportion of a population that is vitamin A sufficient or deficient.
The use of a single question to obtain a woman’s current night blindness status could have underestimated the reported prevalence of night blindness. Additional probing about certain characteristics of night blindness may have caused more women to increase their response to this question. However, it seems likely that women who did not respond to having night blindness may have had a milder form. Thus, those women who did respond to having night blindness were probably affected severely enough to respond in the affirmative. This suggests that our sample should have provided the greatest difference. We could have drawn control blood samples only from women who reported night blindness but passed the NVTT as our comparison group. However, this would have required a much larger sample size and was not feasible given the environment of the current study.
The usefulness of objective measures for program evaluations will be based on their feasibility, cost, sensitivity, and specificity. The NVTT protocol is a very fast test. After dark adaptation, the test takes less than 1 min. Several people can be dark adapted at once and this allows this method to be used for large surveillance and evaluation studies. The NVTT instrument is small and lightweight and does not need an electrical power source. Therefore it can be conducted in any rural area where women can be dark adapted and we have developed a portable dark room for this purpose. The construction of the portable room was described previously (22,23).
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FOOTNOTES |
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2 Supported by a grant from the United States Centers for Disease Control and Prevention (RO8/CCR918936) and funds from the Task Force Sight and Life.
4 Abbreviations used: LED, light-emitting diode; NVTT, night vision threshold test; ROC, receiver operator characteristics; SVA, serum vitamin A; VAD, vitamin A deficiency.
Manuscript received 5 May 2004. Initial review completed 2 June 2004. Revision accepted 2 August 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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