HIV serologically indeterminate individuals: Future HIV status and risk factors.

BACKGROUND: Indeterminate HIV test results are common, but little is known about the evolution of indeterminate serology and its sociodemographic and behavioral correlates. We assessed future HIV serological outcomes for individuals with indeterminate results and associated factors in Rakai, Uganda.
METHODS: 115,944 serological results, defined by two enzyme immunoassay (EIAs), among 39,440 individuals aged 15-49 years in the Rakai Community Cohort Study were assessed. Indeterminate results were defined as contradictory EIAs. Modified Poisson regression models with generalized estimating equations were used to assess prevalence ratios (PRs) of subsequent HIV serological outcomes and factors associated with HIV indeterminate results.
RESULTS: The prevalence of HIV serologically indeterminate results was 4.9%. Indeterminate results were less likely among women than men (adjPR 0.76, 95% CI 0.71,0.81), in unmarried participants than married participants (adjPR 0.92, 95% CI 0.85,99), and in individuals with primary (adjPR 0.90, 95% CI 0.80,1.02), secondary (adjPR 0.83, 95% CI 0.73,0.96) and post-secondary (adjPR 0.75, 95% CI 0.60,0.94) education, relative to no education. The proportions of persons with indeterminate results progressing to HIV positive, negative or indeterminate results in subsequent visits was 5%, 71% and 24%, respectively.
CONCLUSION: HIV serologically indeterminate results were associated with gender and marital status. HIV surveillance programs should develop a protocol for reporting individuals with mixed or persistently indeterminate HIV results on multiple follow-up visits. Most indeterminate results became HIV-negative over time, but follow-up is still needed to detect positive serologies.

Introduction

Enzyme-linked immunosorbent assays (ELISA) or enzyme immunoassay (EIA) are widely used for the detection of HIV infection. Even with HIV rapid diagnostic tests, ELISAs are the preferred method for confirmatory tests [1, 2].

However, two ELISA tests with different antigen or test properties can produce discordant results with one test negative and the other positive, usually termed as discrepant [3, 4], discordant [5-7] or indeterminate [8]. The World Health Organization (WHO) recommends that discordant results should be retested by the two assays initially used and if the results remain discordant, the serum should be considered indeterminate [9].

The causes of indeterminate results range from factors relating to the individual being tested (such as co-infections or cross reactivity with other proteins), test kits being used, assay processing and the population under study [10]. Celum et al. reports that among males, tetanus boosters in the previous two years and having sexual contact with prostitutes were independently associated with HIV indeterminate results; and among females, parity and autoantibodies were independently associated with indeterminate results [11]. HIV indeterminate results have also been associated with systematic lupus erythematosus, rheumatoid factor and polyclonal gammopathy, antibodies to DR-HLA, cross reactivity to core proteins of other retroviruses, mycobacterium leprae infection, heat inactivation of serum samples, in vitro hemolysis, elevated bilirubin levels and tetanus vaccination [12]. An HIV indeterminate result may be associated with acute HIV infection [13-15], and WHO recommends that such an outcome among high risk populations or those with a history of high risk behaviors should be considered as a potential case of acute HIV infection [16].

The prevalence of HIV indeterminate results varies between study populations, assays and test kits [8]. A review of studies using different test kits shows that the prevalence of HIV indeterminate results ranges from approximately 6% to 50% [10]. Other studies report serologically indeterminate prevalence below 6% [6, 8, 17]. However, it is challenging to project future outcomes from existing studies of HIV indeterminate results due to small sample sizes [12-14] and short follow-up time (less than a year) [8, 11, 18]. Understanding the correlates of ELISA indeterminate results may help HIV surveillance programs to select the appropriate tests and to develop a protocol on how to handle individuals with ELISA indeterminate results.

This study used longitudinal data to assess the long-term outcomes and the within person correlation of indeterminate results. We also explored factors associated with HIV indeterminate results.

Methods

Data were derived from the Rakai Community Cohort Study (RCCS), a population-based open cohort in 50 communities in rural Rakai District, Uganda. Following a household census enumeration, all consenting individuals aged 15–49 were interviewed by trained same-sex interviewers. Behavioral (e.g. number of sexual partners, alcohol use, religion), health (e.g. malaria infection), demographic (e.g. age, sex, marital status) and socioeconomic (e.g. education level, occupation) information as well as blood samples for HIV testing were consistently collected across all surveys [19, 20]. The interval between surveys ranged from 12 months to over 18 months.

The present analysis includes 39,440 individuals who provided 115,944 person-visits from 1994 to 2009. HIV was detected by two parallel ELISA test results (See S1 Fig, Supplementary Digital Content 1, which demonstrates the parallel testing algorithm). All HIV testing was done in the same laboratory. The same pair of test kits were used for the first eight surveys. However, a combination of test kits was used for subsequent surveys (for details on test kits used at each survey, their sensitivity and specificity See S3 Table, Supplementary Digital Content 7, which shows the test kits at each round, the sensitivity and specificity and the number of samples tested by each test kit). HIV indeterminate results were defined as discordant ELISA test results (i.e. one positive and one negative). Other variables included in this study were self-reported

Descriptive analysis estimated the proportions of HIV indeterminate results by RCCS visits and covariates of interest. Time-lagged analysis was used to assess the prevalence of subsequent HIV serological outcomes for participants with prior indeterminate, negative or positive HIV serological results among participants with two or more visits (n = 20,000). The HIV status for individuals with two consecutive indeterminate, negative or positive results was further evaluated as a sensitivity analysis. Modified Poisson regression models using generalized estimation equations with robust variance to account for repeated observations were used to examine associations with serologically indeterminate results (for all participants including those with a single visit, n = 39,440). Lorelogram was used to assess the within person correlation of indeterminate results over multiple study visits [21]. A sensitivity analysis was done to assess factors associated with having two or more indeterminate results limited to participants with two or more HIV indeterminate or negative results (n = 20,000). Decision trees were used to determine the trajectory of individuals with HIV negative, positive or indeterminate results at their first visit by tracking their HIV ELISA results for the subsequent three consecutive visits. Associations with a two-sided p-value ≤ 0.05 were considered statistically significant. STATA 14 was used for analysis [22].

The RCCS was approved by Institutional Review Boards (IRBs) in Uganda (The Research and Ethics Committee of the Uganda Virus Research Institute and the Uganda National Council for Science and Technology), and IRBs at Johns Hopkins University and Western IRB. All subjects ≥18 years of age provided written informed consent and minors gave assent with parental/guardian consent to participate in the study. The research was conducted in accordance with the core principles expressed by the Declaration of Helsinki.

Results

There were 115,944 observations from 39,440 participants. The majority (22,326 (57%)) of the participants were female and 17,114 (43%) were male. The prevalence of HIV indeterminate results over all study visits was 4.9% (5,680/115,944); 5.6% (2,760/4,9695) for males and 4.4% (2,920/66,249) for females. The prevalence of indeterminate results varied by survey round (See S3 Fig, Supplementary Digital Content 8, which shows the prevalence of indeterminate results by survey round).

Females were 24% less likely to test indeterminate compared to males (adjPR 0.76, 95% CI 0.71,0.81) (Table 1). The prevalence of indeterminate results was 5.1% (3,694/71,813) and 4.5% (1,986/44,131) for married and unmarried individuals, respectively; and unmarried participants were 8% less likely to test indeterminate compared to married participants (adjPR 0.92, 95% CI 0.85,0.99). The prevalence of indeterminate results among participants with no education was 5.3% (438/8,303), 5.0% (3,802/75,778), 4.5% (1,272/28,037) and 4.4% (168/3,826) for those with primary, secondary and tertiary education, respectively. Compared to participants with no education, those with primary, secondary and tertiary education were less likely to have indeterminate results (adjPR 0.90, 95% CI 0.80,1.02; adjPR 0.83, 95% CI 0.73,0.96 and adjPR 0.75, 95% CI 0.60,0.94, respectively). People who reported not having malaria were more likely to have indeterminate results compared to people who reported having malaria (adjPR 1.23, 95% CI 1.06,1.50). Similarly, people who resided in urban/trading communities were less likely to have indeterminate results compared to people who resided in rural communities (adjPR 0.92, 95% CI 0.85,0.99). The prevalence of indeterminate results was not associated with number of sexual partners and age (Table 1). When the analysis was limited to the first eight rounds, where the testing was performed with the same pair of test kits, the same inferences held (See S4 Table, Supplemental Digital Content 9).

Table 1

Factors associated with HIV serologically indeterminate results among 39440 (115944 person-visits) RCCS participants in Rakai, Uganda (1994–2009).

Factors	Observations (%)	EIA Indeterminatei prevalence	UnadjPR (95% CI)	AdjPR (95% CI)*
Marital Status
Married	71813(62)	3694/71813 = 5.1%	1.00	1.00
Not married	44131(38)	1986/44131 = 4.5%	0.89(0.84,0.95)	0.92(0.85,0.99)
Gender
Male	49695(43)	2760/49695 = 5.6%	1.00	1.00
Female	66249(57)	2920/66249 = 4.4%	0.79(0.74,0.84)	0.76(0.71,0.81)
Education
No education	8303(7)	438/8303 = 5.3%	1.00	1.00
Primary	75778(65)	3802/75778 = 5.0%	0.95(0.84,1.07)	0.90(0.80,1.02)
Secondary	28037(24)	1272/28037 = 4.5%	0.87(0.76,0.99)	0.83(0.73,0.96)
Tertiary	3826(3)	168/3826 = 4.4%	0.80(0.65,99)	0.75(0.60,0.94)
Resident
Rural	70759(61)	3665/70759 = 5.2%	1.00	1.00
Urban/trading	45185(39)	2015/45185 = 4.5%	0.84(0.79,0.90)	0.92(0.85,0.99)
No. sex partners
0	19654(17)	861/19654 = 4.4%	1.00	1.00
1	74904(65)	3625/74904 = 4.8%	1.09(1.01,1.19)	1.01(0.92,1.11)
2	14750(13)	812/14750 = 5.5%	1.24(1.12,1.37)	1.03(0.92,1.15)
3	4227(4)	237/4227 = 5.6%	1.27(1.10,1.48)	1.04(0.89,1.22)
4	1099(1)	53/1099 = 4.8%	1.12(0.85,1.48)	0.92(0.70,1.21)
5+	1310(2)	92/1310 = 7.0%	1.54(1.23,1.92)	1.21(0.97,1.51)
Age
15–19	21876(19)	986/21876 = 4.5%	1.00	1.00
20–24	25266(22)	1242/25266 = 4.9%	1.09(1.00,1.19)	1.00(0.91,1.10)
25–29	23321(20)	1162/23321 = 5.0%	1.10(1.00,1.20)	0.96(0.87,1.07)
30–34	17439(15)	821/17439 = 4.7%	1.05(0.95,1.16)	0.91(0.81,1.02)
35–39	12425(11)	627/12425 = 5.1%	1.15(1.04,1.29)	0.99(0.87,1.11)
40–49	15617(13)	842/15617 = 5.4%	1.21(1.09,1.34)	1.02(0.91,1.15)
Malaria
Yes	3552(3)	112/3552 = 3.2%	1.00	1.00
No	112392(98)	5568/112392 = 5.0%	1.45(1.21,1.73)	1.23(1.06,1.50)

*Model also adjusted for occupation, religion, survey round and region of residence, EIAJ = Enzyme-linked Immunoassay indeterminate.

We performed a similar analysis for factors associated with multiple (two or more) indeterminate results. The overall prevalence of two or more HIV serologically indeterminate results was 6.5% (5,712/8,7945), with 7% (3,822/38,520) for males and 6% (2,890/49,425) for females (adjPR 0.85, 95% CI 0.73,0.98).

Compared to married participants, those not married were less likely to have two or more indeterminate results (adjPR 0.85, 95% CI 0.73,0.98). Similarly, participants who reported not having malaria were more likely to have two or more indeterminate results (adjPR 1.32, 95% CI 1.07,1.64). The prevalence of having two or more indeterminate results was 20% lower comparing participants who reside in urban/trading areas to participants who reside in rural communities (adjPR 0.80, 95%CI 0.68,0.96). A dose response relationship of repeat indeterminate results was observed among age groups. Compared to younger participants, older individuals are more likely to have two or more indeterminate results. The prevalence of having two or more indeterminate results was not associated with other sociodemographic or behavioral factors (See S1 Table, Supplementary Digital Content 2, which shows factors associated with having two or more indeterminate results).

Prevalence of indeterminate results by number of visits

Table 2 shows the distribution of indeterminate results by number of visits per participant. Of the 15,896 individuals with one visit, 4% (636/15,896) had indeterminate results. The frequency of having at least one indeterminate result increased with the number of visits per participant. For example, 11% (536/4,725) of individuals with three, 23% (381/1,689) for individuals with six, 31% (264/856) for those with 9 visits. Finally, for those individuals with 11 visits 35% (149/427) had at least one indeterminate result.

Table 2

Number of EIA indeterminate results by total number of visits per participant.

	Total number of visits
# (D)a	1	2	3	4	5	6	7	8	9	10	11
0	15260	7101	4188	2360	1743	1308	979	750	592	580	278
1	636	499	440	366	302	288	212	210	173	183	87
2		81	70	63	89	58	61	57	49	33	35
3			26	19	27	21	24	22	24	24	14
4				3	5	12	10	10	6	12	6
5					0	2	8	1	9	7	3
6						0	0	3	3	3	2
7							0	0	0	0	1
8								0	0	0	1
9									0	0	0
Total # of obs.b	15896	15362	14175	11244	10830	10134	9058	8424	7704	8420	4697
Total # of indiv.c	15896	7681	4725	2811	2166	1689	1294	1053	856	842	427

a Number of indeterminates

b total number of observations

c total number of individuals.

Within person correlation of indeterminate results

Fig 1 is a Lorelogram which measures the within person correlation of indeterminate results using log odds ratios [21]. Fig 1 suggests that the within person correlation of indeterminate results is autoregressive with individuals being more likely to test indeterminate closer in time to a prior indeterminate result.

Fig 1

Lorelogram.

The x-axis is the time-lag between two measurements and the y-axis is log odds ratio.

Future HIV outcomes for individuals with indeterminate results

We evaluated the probability of transitioning from an indeterminate status to either positive, negative or remaining indeterminate. The proportions transitioning from having an indeterminate result to HIV positive, negative, or remaining indeterminate in subsequent visits were 5%, 71%, and 24%, respectively (Table 3). Participants with indeterminate results who reported having malaria were more likely to transition to HIV positive in subsequent follow-up visits (7/75 = 9%) compared to participants with indeterminate results who reported not having malaria (184/3,852 = 5%). Truck drivers and bar attendants, brewers and hairdressers with indeterminate results were more likely to transition to HIV positive results in subsequent follow-up visits (2/16 = 13% and 4/49 = 8%, respectively) compared to participants in other occupations such as government and salaried employees (11/257 = 4%), students (7/400 = 2%) and agricultural workers (103/2121 = 5%). Similarly, participants with tertiary education who have indeterminate results were less likely to transition to HIV positive in subsequent follow-up visits (6/201 = 3%) compared to participants with primary (128/2,638 = 5%), secondary (41/777 = 5%) or no education (15/310 = 5%). Younger participants (15–19 years) with indeterminate results were less likely to transition to HIV positive in subsequent follow-up visits (8/370 = 2%) compared to participants in older age categories who have indeterminate results (20–24 years 44/813 = 5%; 25–29 years 48/901 = 5%; 30–34 years 35/656 = 5%).

Table 3

The prevalence of transitioning from HIV negative, positive or indeterminate to HIV negative, positive or indeterminate in subsequent follow-up serological tests.

		HIV+	HIV-	Indeterminate
Lagged result EIA
Negative	63743 (83)	1453/63743 = 2.3%	5934/63743 = 93.1%	2949/63743 = 4.6%
Indeterminate	3927 (5)	191/3927 = 4.9%	2779/3927 = 70.8%	957/3927 = 24.4%
Positive	8834 (12)	8611/8834 = 97.5%	116/8834 = 1.3%	107/8834 = 1.2%

a Number of observations, b EIA = enzyme immunoassay.

Individuals who tested indeterminate two or more times were more likely to continue to test indeterminate compared to people who had only tested indeterminate once. The percentage transitioning from two consecutive HIV indeterminate results to HIV positive, negative or indeterminate in subsequent visits were 5%, 50% and 46%, respectively. For comparison, for individuals with two consecutive negative results, the proportions transitioning to HIV positive, negative or indeterminate in subsequent visits were 2%, 93% and 5%, respectively. For participants with two consecutive HIV positive results, the proportions transitioning to HIV positive, negative or indeterminate in subsequent visits were 99%, 0.2% and 0.5%, respectively. (See S2 Table, Supplementary Digital Content 3, which shows transitions from two consecutive negative, positive or indeterminate results to HIV positive, negative or indeterminate in subsequent visits).

HIV serological results trajectory for participants with indeterminate, negative and positive EIA results at their first visit

Of the 39,440 participants in the study 1,667 (4%), 32,867 (83%) and 4,906 (12%) had indeterminate, negative and positive HIV test results at their first visit, respectively (See S2 Fig, Supplementary Digital Content 4, which shows the HIV EIA results at first visit). The HIV results for the subsequent three consecutive follow-up visits for each of these initial serological outcomes at the first visit is illustrated in Fig 2 and Supplementary Digital Content 5 and 6 (S3 and S4 Figs, respectively).

Fig 2

Future HIV EIA results for individuals with indeterminate results at their first visit.

Of the 1,667 participants with indeterminate results at their first visit who had two or more visits (1,031), 22% (229), 72% (744) and 6% (58) had HIV indeterminate, negative and positive results, respectively, at their second visit. Of the 229 participants with indeterminate results at their first and second visit who had three or more visits (148), 43% (64), 50% (74), and 7% (10) had HIV indeterminate, negative and positive results at their third visit, respectively (Fig 2). The trajectory for individuals with positive or negative results at their first visit is illustrated in S3 and S4 Figs (see Supplementary Digital Content 5 and 6, which shows the follow-up serological results for participants with positive or negative HIV results at their first visit). Overall, individuals who tested negative or positive at their first visit were more likely to remain negative or positive at subsequent visits, compared to participants with indeterminate results at their first visit.

Final HIV status for indeterminate observations

Based on past HIV status and additional confirmatory HIV testing, the final HIV status was determined for each indeterminate observation. Of the 5,680 HIV indeterminate results, 95% (5,411/5,680) were determined to be from HIV negative individuals, and 4% (205/5,680) were determined to be HIV positive individuals. However, the HIV status for the remaining 1% (64/5,680) could not be determined even with additional HIV testing.

Discussion

We estimate that the overall prevalence of HIV serologically indeterminate based on parallel ELISA testing results in the Rakai Community Cohort Study was 4.9%. We found that indeterminate results were correlated within individuals and that about a quarter of individuals with an initial indeterminate result will have an indeterminate result a year or more later. The proportions having two or more indeterminate results increased with increased age. However, this may reflect a cohort effect: as people get older, they are more likely to participate in multiple surveys. We cannot disentangle the age effect from testing multiple times. The autoregressive within person correlation of indeterminate results, presented in Fig 1, imply that individuals are more likely to test indeterminate closer in time to a prior indeterminate result. To the best of our knowledge, there are no guidelines on how to handle individuals with persistent indeterminate results or mixed results on multiple visits. It is important for specific guidelines to be developed for such individuals. To date, molecular-based tests such as PCR are commonly used to try to resolve discordant HIV ELISA results, which can sometimes give inconclusive results. Also, PCR may not be feasible in remote, hard to reach settings in low- and middle-income countries [23].

The frequency of indeterminate ELISA results was relatively high compared to other studies [4, 7] but comparable to Western Blot indeterminate prevalence [10]. The frequency of transitioning from indeterminate to negative in subsequent visits agrees with findings from other studies that suggest that individuals with indeterminate results should be considered HIV negative if they are not within a high risk group [8, 11, 24–26]. However, the prevalence of transitioning from indeterminate to HIV positive in subsequent visits is not negligible (~5%), which suggests that individuals with indeterminate results may be in the early phase of infection, as suggested by other studies [13-15]. WHO advises that individuals with indeterminate results, particularly in high incidence settings, may be in the acute phase of infection and should receive follow-up testing [16]. A study by Boeras et al. in Rwanda and Zambia reported that 5% of individuals with indeterminate results seroconverted, similar to the result of this study [3].

The findings from this study suggest that individuals with indeterminate results can remain indeterminate for multiple follow-up visits and their HIV status may not be resolved even with years of follow-up. A study by Meles et al. reports that ~94% of individuals who were initially indeterminate later tested negative during follow-up visits; however, ~7% remained indeterminate after repeated follow up tests. In this study, among participants with three consecutive indeterminate results, ~47%, had another indeterminate EIA result at their fourth visit. This suggests that individuals with persistent indeterminate results are more likely to remain indeterminate. Some of the factors (systematic lupus erythematosus, rheumatoid factor and polyclonal gammopathy, antibodies to DR-HLA, cross reactivity to core proteins of other retroviruses, mycobacterium leprae infection, in vitro hemolysis, parity and tetanus vaccination [12]) known to be associated with indeterminate results are chronic or permanent which in part may explain why certain people may remain indeterminate for a long period of time.

Females, unmarried participants, and individuals with at least primary education were less likely to have indeterminate serologic results. Other studies have demonstrated similar factors correlated with HIV indeterminate results. Carneiro-Proietti et al. reported that 73% of individuals with indeterminate results were male and 54% of all HIV indeterminate individuals were married. Married women in Rakai are less likely to use contraceptives and more likely to have children [27, 28] and parity has been found to be associated with having indeterminate results among females [11]. False HIV EIA positive results has also been reported to be associated with multiparous women [29]. Other factors previously found to be associated with indeterminate serologic results include tetanus immunization in males [11]. However, the prevalence of tetanus immunization among men in Rakai is very low (~23%) [30]. In Uganda, males have higher prevalence of smoking compared to females [31] and it is well established that cigarette smoking can alter blood viscosity [32]. Viscous blood samples or samples with precipitates can form residues which could interfere with ELISA assays [6]. This, in part, may explain why males have higher prevalence of indeterminate results compared to females and it is important for public health screening programs to consider this when screening males for HIV infection.

There are limitations to this study. Test kits that used whole viral lysates are known to have high false positive results and we were not able to stratify our analysis by the antigens used by each test kit [33, 34]. The results presented in this analysis may not hold for other populations, especially those with lower HIV prevalence. In lower prevalence populations, samples with indeterminate results would have a much lower frequency of being true HIV positive. Self-reported malaria infection may not be accurate, and we did not test for other health conditions that have been reported to cause indeterminate results. Additionally, the results from this study should be interpreted with caution given the changes in the HIV epidemic and testing tools over the years. The large sample could also result in statistical significance. Notwithstanding these limitations, this study has significant strength, such as the use of a longitudinal data with parallel ELISA tests done consistently for over 10 years.

The findings from this study are applicable whenever ELISAs are used either for HIV surveillance or clinical diagnoses purposes. In conclusion, females, unmarried individuals and having at least primary education are associated with lower indeterminate results. Individuals with indeterminate results are likely to be HIV negative, but an important proportion (~5%) are found to be HIV positive in subsequent visits. The findings from this study support the WHO recommendation that HIV surveillance programs should analyze and report indeterminate results separately to avoid the over or underestimation of HIV prevalence [9].

Supplemental Digital Content 1: HIV EIA parallel testing algorithm.

(DOCX)

Click here for additional data file.

Supplemental Digital Content 4: HIV EIA results at first visit.

(DOCX)

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Supplemental Digital Content 5: Future HIV EIA results for individuals with HIV negative results at their first visit.

(PDF)

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Supplemental Digital Content 6: Future HIV EIA results for individuals with HIV positive results at their first visit.

(PDF)

Click here for additional data file.

Supplemental Digital Content 8: Prevalence of HIV EIA indeterminate results for RCCS participants in Rakai, Uganda (1994–2009).

(PDF)

Click here for additional data file.

Supplemental Digital Content 2: Factors associated with having two or more HIV serologically indeterminate results among 20000 (87945 person-visits) RCCS participants in Rakai, Uganda (1994–2009).

(DOCX)

Click here for additional data file.

Supplemental Digital Content 3: The prevalence of transitioning from HIV (NN), (PP) or (DD) to HIV negative (N), positive (P) or indeterminate (D) in subsequent follow-up serological tests.

(DOCX)

Click here for additional data file.

Supplemental Digital Content 7: Test kit used for each survey round and the percentage of samples tested by each test kit.

(DOCX)

Click here for additional data file.

Supplemental Digital Content 9: Factors associated with HIV serologically indeterminate results among 26375 (62148 person-visits) RCCS participants in Rakai, Uganda (1994–2002).

(DOCX)

Click here for additional data file.

3 Oct 2019

PONE-D-19-22579

HIV serologically indeterminate individuals: future HIV status and risk factors

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Reviewer #1: Indeterminate test results remain a bane of many within the HIV diagnostics testing arena. While this phenomenon is often accepted as a consequence of testing biological specimens, this manuscript has attempted to quantify the impact of this phenomenon using a statistical lens. While very interesting, this reviewer has concerns re: methodology used from the perspective of the test kits used:

1. The number (6) and nature of the tests used in the 13 phases is a bit concerning as far as generalizability is concerned. They are not consistent and while sensitivity remains 100% the specificity does not. The Welcozyme EIA used in the later phases has 99 % specificity. Considering the range of specificities reported by the different manufacturers, this parameter would be expected to have a potentially significant impact on indeterminate (false-reactive) test results.

2. Because of this can the authors stratify their analysis so that the same EIAs used in the parallel testing algorithm are consistent between phases. By admission the authors state that the same pair of EIAs were used in the first 8 phases. Emphasis should be placed on this phase of the test results to avoid generalizing with later phases that employed different EIAs.

3. The stratification should also distinguish the antigens used between the different EIAs. As an example any EIA employing whole-viral lysate will likely increase false-reactives while those employing synthetic peptides/recombinant antigen would be expected to reduce false-reactives. The discussion should also include this variation between the different EIAs as a limitation to their analysis.

4. Regarding generalizability the positive predictive value (PPV) of a screening test/algorithm will be higher in a high-prevalence population. It would be helpful if the authors could consider a sensitivity analysis on what the impact would be in lower-prevalence population such as North America.

5. Lines 265-269. While the authors make the statement that guidelines are lacking on persistently indeterminate (serology) test results, they should make reference to the fact that molecular-based tests remain a commonly used testing strategy to help resolve these problematic specimens.

6. Line 256. Spelling/grammar; determine(d), the authors should do a better spellcheck as the manuscript is peppered with spelling errors.

7. Line 147. There is an interesting finding re: lower indeterminate result in those residing in urban/trading vs rural residence. Could the authors speculate on why this may be ?

**********

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Reviewer #1: No

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16 Jan 2020

The Editors, PLOS ONE

Editorial Office

November 26, 2019

Dear Editor,

On behalf of my co-authors, I resubmit the enclosed manuscript under the new title, “HIV serologically indeterminate individuals: future HIV status and risk factors” to be considered for publication in PLOS ONE. We thank the editors and reviewers for their valuable comments and suggestions. We provide an edited and clean version of the manuscript. We believe the present manuscript is greatly improved and we thank you for considering our work for publication.

Below is a point-by-point address of our responses to the reviewers.

Sincerely,

Oliver Laeyendecker

Oliver Laeyendecker MS, MBA, PhD

Staff Scientist, NIAID, NIH

Assistant Professor of Medicine, SOM, JHU

Assistant Professor of Epidemiology, JHSPH

855 North Wolfe St.

Rangos Building, room 538A

Baltimore MD, 21205

Phone: 410-502-3268

Email: olaeyen1@jhmi.edu

Editor’s comment . Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, as your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Response: We thank you for drawing our attention to this important information. The ethics statement has been revised as follows “All subjects ≥18 years of age provided written informed consent and minors gave assent with parental/guardian consent to participate in the study.”

Review Comments to the Author

Reviewer #1: Indeterminate test results remain a bane of many within the HIV diagnostics testing arena. While this phenomenon is often accepted as a consequence of testing biological specimens, this manuscript has attempted to quantify the impact of this phenomenon using a statistical lens. While very interesting, this reviewer has concerns re: methodology used from the perspective of the test kits used:

The number (6) and nature of the tests used in the 13 phases is a bit concerning as far as generalizability is concerned. They are not consistent and while sensitivity remains 100% the specificity does not. The Welcozyme EIA used in the later phases has 99 % specificity. Considering the range of specificities reported by the different manufacturers, this parameter would be expected to have a potentially significant impact on indeterminate (false-reactive) test results.

Response: We thank the reviewer for their comment and are addressing these issues below.

2. Because of this can the authors stratify their analysis so that the same EIAs used in the parallel testing algorithm are consistent between phases. By admission the authors state that the same pair of EIAs were used in the first 8 phases. Emphasis should be placed on this phase of the test results to avoid generalizing with later phases that employed different EIAs.

Response: To determine how the change in test kit over time might impact our results, we did a sensitivity analysis limiting the study to round 1 through round 8 where the same pair of test kits were used. The inference did not change when we limited the analysis to these rounds (See supplementary Table 4., Supplemental Digital Content 9). We added the following sentence to the end of the second paragraph of the results section, “When the analysis was limited to the first eight rounds, where the testing was performed with the same pair of kits, the same inferences held (See supplementary Table 4., Supplemental Digital Content 9).”

3. The stratification should also distinguish the antigens used between the different EIAs. As an example any EIA employing whole-viral lysate will likely increase false-reactives while those employing synthetic peptides/recombinant antigen would be expected to reduce false-reactives. The discussion should also include this variation between the different EIAs as a limitation to their analysis.

Response: We thank the reviewer for drawing our attention to this important point. We have added the antigens for each test kits into the supplemental digital content 7, which now shows the test kit, the manufacturers reported sensitivity and specificity and the antigens as well. Due to the nature of the testing we are not able to stratify our analysis by test kit antigens. This will be taken into consideration in future studies when it is possible to stratify by antigens.

In addition, we have added the following sentence to the limitations “Test kits that used whole viral lysates are known to have high false positive rate and we were not able to stratify our analysis by the antigens used by each test kit.”

4. Regarding generalizability the positive predictive value (PPV) of a screening test/algorithm will be higher in a high-prevalence population. It would be helpful if the authors could consider a sensitivity analysis on what the impact would be in lower-prevalence population such as North America.

Response: Unfortunately, we do not have a low prevalence population to directly compare our results to. That said, as the proportion of false positives would increase for each test in a lower prevalence population, the proportion of indeterminate results from truly in infected individuals would increase. We have added the following sentence to the limitations paragraph in the discussion. “The results presented in this analysis may not hold for other populations, especially those with lower HIV prevalence. In lower prevalence populations, samples with indeterminate results would have a much lower frequency of being true HIV positive.”

5. Lines 265-269. While the authors make the statement that guidelines are lacking on persistently indeterminate (serology) test results, they should make reference to the fact that molecular-based tests remain a commonly used testing strategy to help resolve these problematic specimens.

Response: We thank the reviewer for the suggestion. We have added a sentence to the first paragraph of the discussion that states: “To date, molecular-based test such as PCR are commonly used to try to resolve discordant HIV ELISA results, which sometimes can still give inconclusive results. Also, PCR may not be feasible in remote hard to reach settings in low and middle income countries.”

6. Line 256. Spelling/grammar; determine(d), the authors should do a better spellcheck as the manuscript is peppered with spelling errors.

Response: We thank the reviewer for their careful reading of the paper and have made corrections to the document.

7. Line 147. There is an interesting finding re: lower indeterminate result in those residing in urban/trading vs rural residence. Could the authors speculate on why this may be?

Response: We speculate that people who reside in rural communities in Uganda are mostly farmers who are exposed to tetanus (from farm related injuries) and other infections as a result of their farming activities. Such individuals’ immune systems would be more exposed to a broad array of infections and likely generate more cross-reactive antibodies. This in part may explain why we see high prevalence of indeterminates among people in rural areas compared to people who reside in urban/trading areas.

Submitted filename: Response to reviews 11-26-2019.docx

Click here for additional data file.

8 Jun 2020

PONE-D-19-22579R1

HIV serologically indeterminate individuals: future HIV status and risk factors

PLOS ONE

Dear Dr. Laeyendecker,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 23 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Zixin Wang, PhD.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: This paper is quite interesting and presents an interesting phenomenon worthy of publication and reflection. Some significant gaps, however, likely first need address.

Major comments

1. This issue of indeterminate results seems focused on EIA and ELISAs; however, both of those test types are very rarely used for clinical management of patients. Instead RDTs are used. It would be helpful to understand and discuss if this might be a phenomenon for RDTs in their current contexts. Further, it wasn’t entirely clear whether the focus and suggested consideration of these results should be primarily or solely in surveillance studies or actual clinical management and diagnosis.

2. It would be very helpful to indicate somewhere (partly in the abstract, but definitely in the methods) the variety of different behavioral, health, demographic and socioeconomic information collected from each patient. Also, whether this was consistently collected across time points.

3. Some of the more important references provided throughout the introduction are incredibly old (+10 years) and should be updated or else the implications lightened. For example, the WHO recommendation is from 1997 and unlikely to still exist and be relevant. This study itself was done between 1994 and 2009, ending over 10 years ago. This absolutely must be included as a limitation and perhaps clearly and specifically discussed with reference to the results – are/can they still be considered valid given the significant changes in policy and the epidemic since then. Further, it’s unclear if the patients included were known HIV-infected and whether on treatment. If not, that changes the implications of the results given the population of HIV-uninfected and untreated is getting increasingly smaller.

4. In the introduction, a number of similar previous works are referenced; however, neither there nor more importantly in the discussion are these discussed and the importance of the current work to that body touched on.

5. There were several interesting findings, many of which were identified previously, such as males and those married were at higher risk of indeterminate rates. However, this should be discussed further. Why might these issues be happening in those populations?

6. It would also be helpful to understand if any combinations of the factors may result in more indeterminate results. For example, older married males or need those factors always be disaggregated?

7. I’m not sure lines 219-238 bring a lot to the results given all others. I might suggest removing, particularly to include a possible analysis per point #6.

8. It would also be helpful to more clearly discuss possible reasons for why indeterminate results may be persistent. What could be causing this? Lines 272-279. Also, what can or should be done for those patients?

9. It also might be worth reflecting on the large sample size. No sample size calculation was included and though some of the differences are relatively small, the incredibly large sample size would cause significance.

Minor comments

1. Line 36: ‘serological’ results rather than ‘serologic’. Also, ‘immunoassays’ is likely the best terminology.

2. The suggestion that those with primary education are less likely to have indeterminate results should be more carefully considered given the adjPR crosses 1.

3. Line 58, ‘assay’ should likely be ‘assays’ for both.

4. Lines 189-206 should be double-spaced.

5. Line 259-260: I’m not sure that’s still the case given significant scale-up of HIV viral load testing and tuberculosis testing, both molecular PCR-based.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Jul 2020

Dear Editor,

Thank you for the opportunity to revise and resubmit our manuscript, HIV serologically indeterminate individuals: future HIV status and risk factors,” for consideration for publication in the PLOS ONE journal. Please find below a point-by-point response to the reviewers’ comments and corresponding changes made to the manuscript. We have revised the manuscript incorporating nearly all of the reviewer’s recommendations and agree that these revisions have strengthened the quality of the article. Please let us know if we can be of any additional assistance in the review of our manuscript.

Thank you again for your consideration.

Reviewer #1: (No Response)

Reviewer #2: This paper is quite interesting and presents an interesting phenomenon worthy of publication and reflection. Some significant gaps, however, likely first need address.

Major comments

1. This issue of indeterminate results seems focused on EIA and ELISAs; however, both of those test types are very rarely used for clinical management of patients. Instead RDTs are used. It would be helpful to understand and discuss if this might be a phenomenon for RDTs in their current contexts. Further, it wasn’t entirely clear whether the focus and suggested consideration of these results should be primarily or solely in surveillance studies or actual clinical management and diagnosis.

Response: We thank the reviewer for their thoughtful comment. The reviewer is right, the paper is focused on EIA and ELISAs and not on RDTs. Since the paper did not look at RDTs it is impossible to determine if our findings will be applicable to RDTs. ELISAs are used both for surveillance and clinical diagnoses hence we cannot limit our findings to only clinical or surveillance scenarios. In any situation (surveillance or clinical) where EIA and ELISAs are used in the process, our findings may be applicable. In future we plan to examine indeterminate results for RDTs.

We added the following sentence to the manuscript to address this comment “The findings from this study are applicable whenever ELISAs are used either for HIV surveillance or clinical diagnoses purposes.”

2. It would be very helpful to indicate somewhere (partly in the abstract, but definitely in the methods) the variety of different behavioral, health, demographic and socioeconomic information collected from each patient. Also, whether this was consistently collected across time points.

Response: We thank the reviewer for drawing our attention to this important point. The following sentence has been modified to capture both the information collected and the idea that this was consistent over all surveys. We provided reference to earlier publications from RCCs which include details on all the behavioral, health, demographic and socioeconomic information collected from each participant.

“Behavioral (e.g. number of sexual partners, alcohol use, religion), health (e.g. malaria infection), demographic (e.g. age, sex, marital status) and socioeconomic (e.g. education level, occupation) information as well as blood samples for HIV testing were consistently collected across all surveys(1,2).”

3. Some of the more important references provided throughout the introduction are incredibly old (+10 years) and should be updated or else the implications lightened. For example, the WHO recommendation is from 1997 and unlikely to still exist and be relevant. This study itself was done between 1994 and 2009, ending over 10 years ago. This absolutely must be included as a limitation and perhaps clearly and specifically discussed with reference to the results – are/can they still be considered valid given the significant changes in policy and the epidemic since then. Further, it’s unclear if the patients included were known HIV-infected and whether on treatment. If not, that changes the implications of the results given the population of HIV-uninfected and untreated is getting increasingly smaller.

Response: We agree with the reviewer that some of the citations are old and that the data for the study itself is old. However, current information on parallel HIV ELISAs are rare, yet these assays are still used for HIV testing today and the issue of indeterminate results persist. Updated information on HIV ELISA indeterminate results are quite limited in the literature which motivated us to carry out our analysis. We indicated the time period (as correctly reference by the reviewer) when the data for our analysis was collected, the test kits and other details to give readers the opportunity to decide whether our findings will be applicable in their situation.

Treatment became available in Rakai Uganda as part of PEPFAR in 2006. For the time period of this study individuals who had a CD4 count <250 cells/mm3 would be eligible for treatment. When we limited the analysis to the first 8 rounds which occurred between 1994 to 2002 when ART was not available in Rakai, the inference did not change. (See supplementary Table 4., Supplemental Digital Content 9). We did include the following sentence in the manuscript to acknowledge that the data used in the analysis is old. “Additionally, the results from this study should be interpreted with caution given the changes in the HIV epidemic and testing tools over the years.”

4. In the introduction, a number of similar previous works are referenced; however, neither there nor more importantly in the discussion are these discussed and the importance of the current work to that body touched on.

Response: We thank the reviewer for this comment. In the introduction we did indicate the gaps in the existing studies stating that most of the studies have relatively smaller sample size, short follow-up time for people with indeterminate results making it impossible to understand the long-term outcomes of people with HIV indeterminate results. We provided justifications for why we think the current study is important for the advancement of HIV testing.

Throughout the discussion we cited several papers that were initially cited in the introduction section (~8 papers cited in the introduction) to help put our findings in context of the existing literature. For instance, in the discussion we compared the prevalence, correlates and subsequent outcomes of indeterminate results in our study to other studies (citing studies from our introduction). We will be citing additional studies from the studies cited in the introduction to address comment number 5 below.

5. There were several interesting findings, many of which were identified previously, such as males and those married were at higher risk of indeterminate rates. However, this should be discussed further. Why might these issues be happening in those populations?

Response: We thank the reviewer for drawing our attention to this important point. We have added the following sentences to further elaborate on our findings:

“Married women in Rakai are less likely to use contraceptives and more likely to have children(3,4) and parity has been found to be associated with having indeterminate results among females(5). False HIV EIA positive results has also been reported to be associated with multiparous women(6).”

“In Uganda, males have higher prevalence of smoking compared to females(7) and it is well established that cigarette smoking can alter blood viscosity(8). Viscous blood samples or samples with precipitates can form residues which could interfere with ELISA assays(9). This, in part, may explain why males have higher prevalence of indeterminate results compared to females and it is important for public health screening programs to consider this when screening males for HIV infection.”

6. It would also be helpful to understand if any combinations of the factors may result in more indeterminate results. For example, older married males or need those factors always be disaggregated?

Response: We thank the reviewer for the idea of exploring if there is interactive effect. The original goal of this study was not to examine the combined effect of any of the factors. We believe the exploration of the combined effect of multiple indicators and their effect on indeterminate results is a completely different research question which is not a part of the goal of our analysis. This will be considered in future studies.

7. I’m not sure lines 219-238 bring a lot to the results given all others. I might suggest removing, particularly to include a possible analysis per point #6.

Response: Lines 219-238 referenced by the reviewer presents the results for one of the key objectives of the study i.e. the long-term outcomes of people with initially indeterminate results. Removing these lines in our view will defeat the primary goal of the study.

8. It would also be helpful to more clearly discuss possible reasons for why indeterminate results may be persistent. What could be causing this? Lines 272-279. Also, what can or should be done for those patients?

Response: We added some more some more explanations that possibly result in persistent indeterminate results. We added the following sentence to further explain why indeterminate results may be persistent:

“Some of the factors (systematic lupus erythematosus, rheumatoid factor and polyclonal gammopathy, antibodies to DR-HLA, cross reactivity to core proteins of other retroviruses, mycobacterium leprae infection, in vitro hemolysis, parity and tetanus vaccination(10)) known to be associated with indeterminate results are chronic or permanent which in part may explain why certain people may remain indeterminate for a long period of time.”

9. It also might be worth reflecting on the large sample size. No sample size calculation was included and though some of the differences are relatively small, the incredibly large sample size would cause significance.

Response: We thank the reviewer for this comment. The following sentence has been added to the limitations section: “The large sample could also result in statistical significance.”

Minor comments

1. Line 36: ‘serological’ results rather than ‘serologic’. Also, ‘immunoassays’ is likely the best terminology.

Response: We thank the reviewer for thoroughly reading our paper. The above error has been fixed.

2. The suggestion that those with primary education are less likely to have indeterminate results should be more carefully considered given the adjPR crosses 1.

Response: We thank the reviewer for drawing our attention to this point. We were interpreting the point estimate and we were not necessarily referring to statistical significance in this situation.

3. Line 58, ‘assay’ should likely be ‘assays’ for both.

Response: We thank the reviewer for thoroughly reading our paper. The above error has been fixed.

4. Lines 189-206 should be double-spaced.

Response: We thank the reviewer for thoroughly reading our paper. The above error has been fixed.

5. Line 259-260: I’m not sure that’s still the case given significant scale-up of HIV viral load testing and tuberculosis testing, both molecular PCR-based.

Response: We thank the reviewer for their thoughts on this. It is true that there are some advances in scaling up PCR-based HIV viral load and tuberculosis testing, but this is still limited to large central hospitals or labs at the national level in several low- and middle-income countries. We do not believe PCR-based testing is available particularly at the subnational level in several LMICs.

Submitted filename: Rebuttal Letter with responds to reviewers.docx

Click here for additional data file.

31 Jul 2020

HIV serologically indeterminate individuals: future HIV status and risk factors

PONE-D-19-22579R2

Dear Dr. Laeyendecker,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Zixin Wang, PhD.

Academic Editor

PLOS ONE

14 Aug 2020

PONE-D-19-22579R2

HIV serologically indeterminate individuals: future HIV status and risk factors

Dear Dr. Laeyendecker:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Zixin Wang

Academic Editor

PLOS ONE