Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

BACKGROUND: Toxoplasmosis is caused by an obligate intracellular tissue protozoan parasite, Toxoplasma gondii that infect humans and other warm-blooded animals. Transmission to humans is by eating raw or inadequately cooked infected meat or through ingestion of oocysts that cats have passed in faeces. Studies have shown life-threatening and substantial neurologic damage in immunocompromised patients; however, 80% of humans remain asymptomatic. The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in HIV positive patients and the risk factors associated with the infection, and to investigate the correlation between CD4+ T-cell count and toxoplasma specific antibodies as possible predictors of each other amongst HIV patients in the Bamenda Health District of the North West Region of Cameroon.
METHODS: A cross-sectional study was conducted, in which 325 HIV patients were recruited for administration of questionnaire, serological diagnosis of T. gondii and measurement of CD4+ T-cell count. Bivariate and multivariate logistic regression was used to identify risk factors associated with T. gondii infection while the linear regression was used to investigate the relationship between CD4+ T-cell count and antibody levels against T. gondii.
RESULTS: The findings showed that, majority (45.8%) of HIV patients suffered from chronic (IgG antibody) infection, and 6.5% from acute (IgM and IgM/IgG antibody) toxoplasma infection. The overall sero-prevalence of T. gondii infection amongst HIV patients was 50.5%. On the whole, 43 men (45.7%) and 127 women (55%) presented with anti- T. gondii antibodies; however, there was no significant difference amongst males and females who were positive to T. gondii infection (p = 0.131). Marital status (p = 0.0003), contact with garden soil (p = 0.0062), and garden ownership (p = 0.009), were factors that showed significant association with T. gondii infection. There was no significant difference (p = 0.909) between the mean CD4+ T-cell count of HIV patients negative for toxoplasma infection (502.7 cells/mL), chronically infected with T. gondii (517.7 cells/mL) and acutely infected with T. gondii (513.1 cells/mL). CD4+ T-cell count was neither a predictor of IgM antibody titer (r = 0.193, p = 0.401), nor IgG antibody titer (r = 0.149, p = 0.519) amongst HIV patients acutely infected with T. gondii.
CONCLUSION: The findings from this study underscore the need to implement preventive and control measures to fight against T. gondii infection amongst HIV patients in the Bamenda Health District.

Introduction

Toxoplasmosis caused by the obligate intracellular parasite, Toxoplasma gondii (T. gondii) is a globally prevalent zoonotic disease of warm-blooded animals, including man. Within the human population, the infection is clinically silent in immunocompetent host and accounts for focal encephalitis, headache, confusion, motor weakness and fever amongst patients with acquired immunodeficiency syndrome (AIDS) or immuno-compromised patients [1]. In the absence of treatment, the disease progression results in seizures, stupor, coma and finally death [2]. The infection is contracted through consumption of oocyst in contaminated water, food or from inadequately cooked infected meat. Other means of infection include blood transfusion and organ transplantation [3].

Knowledge of T. gondii seroprevalence and its associated risk factors is important to predict the risk of infection in humans. Studies elsewhere have implicated consumption of raw garden produce, level of education and age as risk factors of the infection [4-6].

Furthermore, toxoplasmosis has been described as the commonest cerebral opportunistic infection in HIV infected patients [7] and a common cause of mortality amongst severely immunosuppressed HIV patients [8]. It is known that a CD4+ T-cell count of ≤200cells/mm3 accounts for reactivation of latent infection in HIV patients [9]. It is therefore rational to think that the magnitude of humoral immune response (IgG and IgM) is a function of CD4+ T cell count, since following T. gondii infection, a rise of specific acquired immunity, including humoral immunity accompanied by parasite clearance from the body [10]. If this is true, then high antibody titer in HIV infected toxoplasma patients should positively correlate with CD4+ T cell count, and consequently, either may serve as a marker of the other.

Methods

Study area and setting

The study was carried out in the Bamenda Health District (BHD), located on the High Western Plateau of Cameroon from August to October 2018. Bamenda is an urban setting, which is both the Administrative Headquarter of Mezam Division and the North West Region of Cameroon. The weather is warm and wet in the rainy season, which last from March to October. In the dry season (November to February), the mornings are generally very cold, and the afternoons are very hot compared to the rainy season with an average annual temperature of 19.3°c. This cosmopolitan city is made up of mainly civil servants and business men and a majority of the population is partly involved in small scale farming. The BHD has a population of over 350,000 inhabitants. It comprises of 17 health areas, 14 public, and 4 faith-based health facilities (1 Baptist, 1 Presbyterian and 2 Catholics) with the Bamenda Regional Hospital (public) being the leading health facility. Amongst the 18 health facilities, there are only four HIV treatment centers (Bamenda Regional Hospital, Nkwen Sub-divisional Medicalised Health Center, Nkwen Baptist Hospital and Mezam Polyclinic) in the BHD.

Study design

A cross sectional study was conducted in the Bamenda Health District where data was collected from August to October 2018. Participants were recruited by a convenient sampling technique at the HIV treatment centers of the Bamenda Regional Hospital and the Nkwen Sub-divisional Medicalised Health Center. The Bamenda Regional Hospital being the referral hospital and leading in terms of the HIV/AIDS patient population in the health district was purposively chosen for this study, while the Nkwen Sub-divisional Medicalized Health Center was randomly selected from the other three HIV treatment centers within the health district. Assuming a prevalence of 69.6% from a previous study elsewhere (Yaounde) in Cameroon [11], the sample size was calculated using the formula of Suresh and Chandrashekara [12]. A value of 323 was achieved which was rounded up to 325 during implementation. Three hundred and twenty-five consenting HIV patients 12 years old and above, who were registered at the treatment centers as residents in the BHD were enrolled into the study at the level of the treatment center during a visit. Participants were administered a structured questionnaire and 3 mL of venous blood was collected from each, into a dry tube for serological diagnosis of T. gondii infection, and another 3 mL collected into Ethylenediaminetetraacetic Acid (EDTA) tube for measurement of CD4+ T-cell count.

Administration of questionnaires

Ten questionnaires were pretested at the treatment center of Ndop District Hospital in the North West region of Cameroon in June 2018. After the pretest exercise, the questionnaire was adapted to be completed within 7–10 minutes including keeping the question of age as an open-ended question. Validated questionnaires were administered to participants of all age groups to obtain data on demography (age of participants, gender, marital status, religion, level of education and occupation), risk factors and if participants had any clinical signs and symptoms of T. gondii infection, or a history of the infection. Risk factors investigated in this study were; duration on ARV, history of default to HIV/AIDS therapy, availability of cats in participant’s home or neighborhood, garden ownership and nature of participant’s contact with garden soil, consumption of raw and/or inadequately cooked meat and/or vegetables, source of domestic water supply, and treatment and storage condition of drinking water.

Laboratory analysis

Serological diagnosis of T. gondii infection

Serological screening for Toxoplasma infection was achieved using Aria Toxo IgG/IgM Combo Rapid Test with accuracy of 94.9% and 97.8% IgG and IgM respectively (CTK Biotech, Inc., San Diego California, USA) according to the manufacturer’s instructions [13]. The kit is a lateral flow chromatographic immunoassay; it detects simultaneously the presence of T. gondii IgG and IgM specific antibodies in patient’s blood. Antibodies against T. gondii in patient’s serum, binds to recombinant T. gondii antigen conjugated with colloidal gold and the coloured complex is captured in the test window coated with mouse anti-human antibody. In essence, 10 uL of serum obtained from patient’s blood was applied to the sample pad alongside 70 uL of kit’s diluent and the result was read after 10 minutes.

The amount of T. gondii IgM antibodies was estimated for IgM positive samples using the Biorex diagnostic T. gondii IgM (Antrim, United Kingdom) following manufacturer’s instructions. The T. gondii IgM enzyme immunosorbent assay (EIA) Test kit is a solid phase EIA based on immunocapture of IgM antibodies in patient’s serum. The presence of T. gondii specific antibodies in patient’s serum complexes with added recombinant T. gondii antigen-enzyme conjugate and reveals a coloured product upon addition of substrate. In brief, following addition of 100 uL of negative control, cut-off calibrator and positive control in appropriate wells, 100 uL of specimen diluent and 5 uL of specimen was added to each of the other wells followed by incubation at 37°C for 30 minutes and repeated washing. Except for the blank well, 100 uL of conjugate was added to each well and incubated at 37°C for 30 minutes, followed by repeated washing; after which 50uL of substrate A and 50uL of substrate B were added, and microwell plate was incubated at 37°C for 10 minutes. The reaction was stopped by addition of 50 uL of stop solution and the optical density was read at 450nm and 650 nm within 30 minutes, using the Emax Precision Microplate ELISA reader (Molecular Divices, California, USA).

The amount of IgG antibodies specific for T. gondii in the IgM positive samples was estimated using the Erbalisa® Toxoplasma IgG (Calbiotec, California, USA) according to manufacturer’s instructions. The assay is composed of pre-coated wells with purified T. gondii antigen, to which patient’s serum is added for T. gondii specific antibodies to bind; if present, an enzyme conjugate is added, which releases a coloured product upon addition of substrate. The assay was carried out by dispensing 100 uL of diluted sample, calibrator, controls and diluent or blank into appropriate wells, and incubated at room temperature for 20 minutes, before washing three times. This was followed by addition of enzyme conjugate and incubation was done at room temperature for 20 minutes before washing. One hundred microlitres of TMB substrate was added to each well and incubated at room temperature for 10 minutes before the reaction was stopped using 100 uL of stop solution. The optical density was read at 450 nm and 650 nm using Emax Precision Microplate ELISA reader (Molecular Divices, California, USA).

Measurement of CD4+ T-cell count

The Pima™ Analyser was used (SN; PIMA-A-005920; Alere Technology Gmbh Loebstedter Street 103–105 Jena, Germany), in the measurement of CD4+ T-cell count, which employs a static image analysis. In essence plasma obtained from whole blood collected in EDTA tube was kept at 18°C and analysis was done within 24hrs. Plasma sample was loaded to PIMA CD4+ test cartridge and ran in the analyser following insertion of test cartridge. Results were automatically calculated and absolute T-cell counts displayed.

Ethical considerations

Ethical clearance for this study was obtained from the University of Buea, Faculty of Health Science (FHS) Institutional Review Board (IRB), reference number 2018/2244UB/SG/IRB/FHS and the Bamenda Regional Hospital Institutional Review Board, reference number 26/APP/RDPH/RHB/IRB. Administrative authorization was obtained from the Regional Delegation of Public Health for the North West Region (RDPH), reference number 233/ATT/NWR/RDPH. Only consenting participants from the study population were recruited into the study, and they all signed the informed consent form. For participants below the age of 21 years old, a signed ascent form was also obtained from their parents or guardians to enable them take part in the research. The participant’s selection exercise was completely a random process.

Statistical analysis

The data were analyzed using Microsoft excel 2010 and IBM SPSS version 25 with significance level judged at p<0.05. Descriptive statistics was computed to establish the frequency of each response type in the sociodemographic characteristic and the sero-prevalence in the study population. Independent variables (socioeconomic, demographic and nutritional risk factors) were analyzed using bivariate and multivariate logistic regression to eliminate confounders and ascertain the level of significance as suspected risk factors of T. gondii infection. A one-way ANOVA test was conducted to compare the difference between the mean CD4+ T cell count groups of HIV/AIDS patients who were either T. gondii infection negative, chronic toxoplasma infection or acute toxoplasma infection. Linear regression analysis was computed to establish relationships between IgM and IgG antibody levels against T. gondii with CD4+ T cell count.

Results

The overall seroprevalence of T. gondii in the BHD of the North West Region of Cameroon was 50.5% (CI = 45.3%-55.7%). The prevalence of chronic (IgG antibody) toxoplasma infection was 45.8% and acute (IgM antibody) infection was 6.5%. The ages of the study participants ranged from 12 to 73 years with an overall mean age of 41.83±13.41years. Majority of the participants, 56.9% (185) were above the age of 41 years, while 43.1% (140) were below the age of 41 years. A Pearson Chi-Square test indicated that there was a significant difference in the seroprevalence of T. gondii infection across the age groups of HIV patient χ2 (2) = 8.180, p = 0.017, with older patients having a higher prevalence (Table 1).

Table 1

Prevalence of T. gondii by age, antibodies, and gender in the BHD.

Characteristics	Positive No (%).	Negative No (%)	Total No (%)	χ2	p-value
Antibodies
IgG only	149(45.8)	176(54.2)	325(100)	-	-
IgM only	6(1.8)	319(98.2)	325(100)	-	-
IgG/IgM	15(4.6)	310(95.4)	325(100)	-	-
Gender
Female	127(55.0)	104(45.0)	231(100)	2.283	0.131
Male	43(45.7)	51(54.3)	94(100)	-	-
Age
≤21	10 (3.1)	20 (6.2)	30(9.3)	8.180	0.017
22–40	52(16)	58 (17.8)	110(33.8)	-	-
41≥	108 (33.2)	77 (23.7)	185(56.9)	-	-

% = percentage, χ2 = chi square.

Majority of HIV patients recruited for the study were females 71.1% (231), as compared to males 28.9% (94). One hundred and twenty-seven (55%) females as compared to 43 (45.7%) males were positive for T. gondii infection. A Pearson Chi-Square test analysis indicated that there was no statistically significant difference between the number of male and female HIV patients who were positive for T. gondii infection χ2 (1) = 2.283, p = 0.131.

Risk factors of T. gondii infection in the study population

In the bivariate and multivariate logistic regression analysis, factors that were statistically significantly associated to the development of T. gondii in HIV patients included demographic factors; marital status (p≤0.001) and socioeconomic factors; contact with garden soil (p = 0.006), and garden ownership (p = 0.009).

Demographic, nutritional and socio-economic factors associated with T. gondii infection

T. gondii infection was significantly more prevalent among HIV patients who were Single/Divorced/widow(er) 54.5% (177/325) (Table 2), compared to HIV patients who were married 45.5% (148/325). The risk of developing Toxoplasma infection was about 2.4 times significantly higher among HIV positive patients who were either Single/Divorced/widow(er) (OR = 2.41, CI = 1.49–3.88, p≤0.001), compared to patients who were married.

Table 2

Association of T. gondii infection and demographic, nutritional and socio-economic factors among HIV patients in the BHD.

Variable	Seroprevalence	Risk factors for T. gondii seroprevalence
	No (%)	Bivariate Analysis		Multivariate Analysis
		COR (95%CI)	p-value	AOR (95%CI)	p-value
Marital status
Married	148 (45.5)	1
Single/Divorce/widow(er)	177 (54.5)	1.81 (1.17–2.83)	0.008	2.41(1.49–3.88)	≤0.001
Age
≤21	30 (9.2)
22–40	110 (33.8)	1.79(0.77–4.18	0.176
≥41	185 (57)	2.74(1.22–6.19)	0.015
Occupation
Business	87 (26.8)	1
Farming	93 (28.6)	1.24(0.69–2.23)	0.479
Others	117 (36)	1.05(0.60–1.83)	0.858
Student	28 (8.6)	0.44(0.18–1.09)	0.075
Consumption of raw/poorly cooked meat
No idea	85 (26.1)	1
No	189 (58.2)	1.07 (0.6405–1.7853)	0.798
Yes	51 (15.7)	0.77 (0.3815–1.5366)	0.453
Consumption of raw/poorly cooked vegetable
No	67 (20.6)	1
Yes	258 (79.4)	1.53 (0.8904–2.6310)	0.124
Contact with garden soil
No	84(25.8)	1
Yes	241(74.2)	3.44(2.02–5.87)	<0.001	7.03(1.74–28.79)	0.006
Owns a garden
No	79 (24.3)	1
Yes	246 (75.7)	2.63(1.55–4.46)	≤0.001	2.19(1.21–3.96)	0.009
Owns a cat
No	218 (67.1)	1
Yes	107 (32.9)	1.60(1.00–2.56)	0.049
Neighbour owns a cat
No	71 (21.2)	1
Yes	254 (78.2)	2.07(1.20–3.54)	0.008

COR = Crude odd ratio; AOR = Adjusted odd ratio; % = percentage; CI = Confidence Interval.

In the bivariate logistic regression, no nutritional factor (consumption of improperly cooked or raw meat p = 0.798 and consumption of improperly cooked vegetable or salad p = 0.124) was significantly associated to the development of Toxoplasma infection (Table 2).

The proportion of HIV patients who presented with Toxoplasma infection (Table 2), and had been in contact with garden soil 74.2% (241/325) was significantly higher compared to HIV patients who presented with Toxoplasma infection without contact with garden soil 25.8% (84/325), (p = 0.006). The risk of developing toxoplasmosis was found to be about 7 times significantly higher among HIV patients who had been in contact with garden soil (OR = 7.03, CI = 1.74–28.79, p = 0.006), compared to HIV patients who had no contact with garden soil (Table 2).

Majority of Toxoplasma infected HIV patients owned gardens 75.7% (246/325), compared to those who did not own a garden 24.3% (79/325), (p = 0.009). The risk of developing Toxoplasma infection was found to be about 2 times significantly higher among HIV patients who owned gardens (OR = 2.19, CI = 1.21–3.96, p = 0.009), compared to HIV patients who did not own a garden (Table 2).

Determination of CD4+ T-cell count as a predictor of acute Toxoplasma infection

One-way analysis of variance (ANOVA) test indicated that there was no significant difference (p = 0.909) between the mean CD4+ T-cell count of HIV patients who were negative for toxoplasmosis and those who were chronically infected or acutely infected with T. gondii infection (Fig 1). Toxoplasma infection was not significantly associated with CD4+ T-cell counts ≤200cells/mL (p = 0.536).

Fig 1

Mean CD4+ T-cell counts between different status of Toxoplasma infection.

Toxoplasma negative = participants with neither IgM nor IgG antibodies against T. gondii infection. Chronic Toxoplasma Infection = participants who were positive only for IgG antibodies against T. gondii. Acute Toxoplasma Infection = participants who were at least positive for IgM antibodies against T. gondii infection.

Relationship between IgM antibodies against Toxoplasma infection and CD4+ T-cell count in HIV patients with T. gondii infection

Amongst the 21 patients with acute infection (positive for IgM antibodies to T. gondii, IgM antibody titers decreased with increasing CD4+ T-cell counts (r = 0.193), but there was no statistically significant difference (p = 0.401) in IgM antibody titer at different levels of CD4+ T-cell counts (Fig 2).

Fig 2

IgM antibodies against Toxoplasma infection and CD4+ T cell count in HIV patients.

Relationship between IgG antibodies against Toxoplasma infection and CD4+ T cell count in HIV patients with acute toxoplasmosis

Amongst the 21 HIV patients acutely infected with T. gondii, there was an increase in IgG antibody titers as the amount of CD4+ T cell counts increased, but there was no linear relationship (p = 0.519) as seen in Fig 3.

Fig 3

IgG antibodies against Toxoplasma infection and CD4+ T cell count in HIV patients.

Discussion

Results from this study revealed a Toxoplasma infection prevalence of 50.5% (164/325). This is less than the prevalence reported in other studies where the prevalence of latent toxoplasmosis was 93.3% in Addis Ababa, Ethiopia, [14]; 60% in Muttu Karl Hospital, Gonder, Ethiopia [15]; 96.3% in North Iran [16] and 69.9% in HIV patients attending the University Teaching Hospital in Yaoundé, Cameroon [11]. The high prevalence of chronic Toxoplasma infection (45.8%) relative to acute infection (6.5%) can be explained by the improved HIV treatment package which reduces mortality and the duration of immune suppression in HIV patients, thus prolonging life expectancy and increasing the number of Toxoplasma chronically infected patients.

Toxoplasma infection was found to be significantly associated (p = 0.006) with HIV patients who had contact with garden soil (74.2%) and those who owned gardens (75.7%, p = 0.009). However, the infection was not significantly associated with consumption of raw or inadequately cooked meat (p = 0.45), and consumption of raw or inadequately cooked vegetable (p = 0.124). This finding is contrary to what was obtained in Ethiopia amongst a similar group of patients, where anti-T. gondii seropositivity was significantly associated with raw meat consumption (p = 0.025) [17]. Consumption of raw meat is not a common practice in the BHD as most of the staple dishes in this locality entail rigorous cooking. In this study; age, cat ownership or neighbors owning cat, were not significantly associated with Toxoplasma infection following multivariate analysis. This is contrary to the observation amongst HIV infected women in Ethiopia where the age group 28 to 37 years was a significant risk factor for Toxoplasma infection [18]. In the same light, studies carried out in Lagos, Nigeria showed that Toxoplasma infection was associated with participants living in close proximity with cats (p = 0.013), contradicting the findings from this study [19]. This study also showed that Toxoplasma infection was significantly associated with single/divorce/widow(er) (p≤0.001), which is contrary to observations in Southern Iran where marital status was not a risk factor for the disease [20]. These findings suggest that the epidemiology of T. gondii infection may vary in different settings.

Although other studies [21, 22], have shown that CD4+ T cell count threshold of ≤200cells/mL was indicative of onset of clinical manifestations of toxoplasma infection, in this study, it was observed that there was no significant difference between the mean CD4+ T-cell count of non-infected, chronically infected and acutely infected patients of toxoplasma infection (p = 0.909). The fact that there was no significant correlation between CD4+ T-cell count and IgM antibody titer (r = 0.193, p = 0.401) suggests that the quantity of IgM antibodies elicited in acutely infected patients is independent of the level of CD4+ T-cells present, although a study in South Brazil reported that CD4+ T-cell count ≤350cells/mm3 of blood, was significantly associated (p = 0.001) with development of acute toxoplasmosis [23]. Antibody titers of IgG showed a positive correlation with increasing CD4+ T-cells; however, it was not significant (r = 0.149, p = 0.519). This suggest that CD4+ T-cell count does not differ from one serological status to the other in T. gondii infection. This suggests that IgG antibody titer cannot be used to predict CD4+ T-cell count in Toxoplasma gondii patients presenting with IgG and IgM antibodies.

Conclusion

The prevalence of T. gondii is still high, despite the successes recorded by the package put in place by WHO through the health system to control and manage opportunistic infections among HIV/AIDS patients. These findings therefore have profound clinical and epidemiological significance and call for more urgent action.

Raw data for toxo project.

(XLSX)

Click here for additional data file.

15 Jun 2020

PONE-D-20-04369

Risk factors for sero-prevalence of Toxoplasma infection and the role of CD4+ T-cell count as a determinant of acute infection amongst HIV patients in the Bamenda Health District, Cameroon.

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Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: No

Reviewer #3: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. 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: The manuscript titled "Risk factors for sero-prevalence of Toxoplasma infection and the role of CD4+ T-cellcount as a determinant of acute infection amongst HIV patients in the Bamenda Health

District, Cameroon" was well written and contains good information. But it could not present the results in a tight manner. For example, the demographic, economic, and nutritional variables are related to each other and affected each other and should have been examined together. And it was necessary to determine the basis on which the variable is included for the multivariate examination.

Also, with respect to the numerical variables, the authors did not reveal the nature of the distribution of those variables and whether they followed the normal distribution in order to use Anova test and why not use T-Test?.

In addition, the authors treat the Odd ratio test on the basis that it quantites the association between  the variables and this is incorrect.  Odd ratio quantifies the strength of the association between two events. There are other tests to do this, such as a risk ratio (RR). On the other hand, we cannot say that the risk increases by 2.4 (table 2 etc..), in fact, the increase is only 1.4 because one is not calculated, as it means that there is no difference.

Other minor corrections include:

It would be better for the author to indicate the average temperatures in the study area. For the phrase "Toxoplama infection" throughout the manuscript, the  the genus Toxoplasma should be written in Italic. The age group 40 and more should be corrected (Table 1).

Reviewer #2: This paper analyzed the seroprevalence of toxoplasmosis in HIV-infected patients in the DHB, Cameroon. Then the authors searched for risk factors for toxoplasmosis. The technical part is well described and the paper is well written. However, it suffers from insufficiencies and inadequate data analysis, which flaws the conclusion.

There are several major problems in the understanding of this study. First of all, the level of CD4+ T cells is well-known as a determinant factor favoring Toxoplasma reactivation, it is not “hypothesized” (line 72). Why do the authors think that “it is necessary to determine the relationship between CD4+ T-cell count and antibody titer to current Toxoplasma infection” (line 75-76) to guide patient management? After primary infection, the residual level of specific IgG is very different from one patient to another, whatever their HIV status. This seems the announced goal of the study (which does not make real sense), but despite that, the main part of the paper is dedicated to antibody screening and risk factor analysis.

Regarding the serological analysis, the most faithful seroprevalence should be calculated on the detection of IgG, regardless the presence of IgM. Indeed, the sole presence of IgM does not necessarily means that it is an acute infection, as non-specific IgM can be detected with many assays. As it is a cross-sectional study, we don’t have the result of a serological follow-up confirming primary-acquired infection by the rise of specific IgG. Thus seroprevalence should be 164/325. Besides, it is incorrect to state that IgM-positive patients have “acute” infection (line 185), as IgM can persist for months or years after primary infection (or be non-specific). Only IgG avidity testing would help confirming possible recent infection. What is really missing here is the context of patient inclusion. Where they consulting for routine checkup and delivery of antiretroviral therapy, or because of clinical signs? This would make a difference in the interpretation of serological results. Anyway, as expected, the seroprevalence increases with age, this is no novelty. There is no need to re-analyze age categories in Table 2. Tables 2, 3 and 4 should be merged.

The analysis of CD4+ T cell counts among Toxoplasma-seropositive and Toxoplasma-seronegative patients does not make sense. Do the authors expect that it influences the contamination with the parasite, which is environmental and foodborne ? it only influences the reactivation of past infection, which is not addressed here. Not surprisingly, there is no correlation between IgG or IgM titers and CD4+ cell counts. Comments about a trend (lines 248-51 and 255-57) are overstated and should be removed.

In the Discussion, it would be interesting to focus on prevalence data obtained in other African countries on the same patient population (HIV+), and to search for possible explanations for this discrepancy with previous results from Cameroon: is the DHB particular in terms of demography, climate, cultural behavior…? This part is well discussed, but the setting of the studies used for comparison should be more precisely addressed (HIV? Pregnant women? Randomly selected population?).

The sentence: “Contrary to other studies, which have shown that CD4+ T cell count threshold of ≤200cells/mL was indicative of onset of clinical manifestations of toxoplasma infection, in this study, it was observed that there was no significant difference between the mean CD4+ T-cell count of non-infected, chronically infected and acutely infected patients of toxoplasma infection” demonstrates that the authors poorly understand the pathophysiology of the disease and mix up serological and clinical findings. Again, the population study (not described here) is probably not the same as the studies cited. Most patients included, I guess, had no clinical signs and visited the health care facility for routine checkup, not because of clinical signs. Additionally, the authors considered that the diagnosis relied on their serological results (i.e. IgG+ = chronic infection, IgM+= acute infection), which is perfectly wrong. Indeed, acute toxoplasmosis in HIV+ patients mostly results from reactivation, thus all patients with clinical signs AND specific IgG (NOT IgM) should considered at risk for reactivation.

Altogether, this paper relies mainly on a seroprevalence study and associated risk factors and should only present the related results.

Reviewer #3: This study has been done to evaluate the seroprevalence of T. gondii infection in 325 HIV positive patients and the risk factors associated with the infection. Authors investigated the threshold of CD4+ T- cell count associated with acute T. gondii infection in the Bamenda Health District of the North West Region of Cameroon.

Comments to authors:

Introduction

Line 58: please write the full scientific name, Toxoplasma gondii, when you mention the name for the first time in the manuscript

Lines 67-69: it would be useful to present data regarding the seroprevalence of T. gondii from different areas of the world, to better put this study in the big picture, and to make this study more interesting to international readers.

Line 69: reference 4 is a poster presentation, please provide full length papers as references

Line 71: reference 7 is a poster presentation of a case report. Authors should provide full length papers as references

Lines 99-100: authors to rephrase the sentence, it is not clear

Lines 120-153: Methods, Serological diagnosis of T. gondii infection, sensitivity and specificity would be good to provide for the serological tests

Lines 120, 128, 130, 179, 198, 203, 204, 206, 212, 216, 219 aso in the paper: “Toxoplasma infection”, please use full scientific name in scientific writing, “T. gondii infection”

Line 200: please write toxoplasmosis, with lower case t

Lines 320-321: the journal is not BMC Veterinary Research, please check and provide journal’s name

Line 377, list of abbreviation: “Toxoplasmosis gondii”, please write “Toxoplasma gondii”

**********

6. 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

Reviewer #3: 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.

11 Jan 2021

Response to Reviewers.

Dear reviewers kindly fine in this later the response to the questions posted.

The topic of the manuscripts has been modified to “Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon” to tie the research with the scientific outcome. the map have been removed because it does not significantly add value to the article. Information about the pretest procedure of the question is as described in line 124 in the questionnaire administration sub heading.

During analysis, the numeric variables followed an abnormal distribution and the analysis of variance test was used.

Determining the relationship between CD4+ T-cell count to antibody titer was to evaluate the effect of the parasite on the immune system in the study population. The investigator captured the aspect of risk factors to ascertain the epidemiology situation of the parasite in the study population. The investigator used a highly specific and sensitive screening test to ascertain the status of infection of the parasite among the study population. It is also true that, the presence of IgM antibodies is the indication of onset of infection and/or recontamination. The inclusion criteria have been addressed in the revised manuscript line 119 indicating the patients were for their routine drug pick up.

The analysis of CD4+T-cell counts among toxoplasma negative and positive patients was to proof the hypothesis that say immune depression (Low CD4+ T-cell count) futher puts the patients in danger to Toxoplasmosis complications.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

24 Mar 2021

PONE-D-20-04369R1

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

PLOS ONE

Dear Dr. Enah,

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.

The authors write the article in a good way. However, they need to address some concerns related to the statistical analysis as requested by reviewer 1. Also, they need to enhance the discussion part. The results contradict most of the previously published articles. Thus, the authors need to discuss the results critically. There are other minor corrections required to be addressed to enhance the quality of the article.

Please submit your revised manuscript by May 08 2021 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:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohammed Abdelfatah Mosa Alhoot, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Authors write the article in good way but need to address the some concerns related to the statistical analysis as requested by the reviewer 1. Also they need to enhance the discussion part. The results come in contradiction with most of previously published articles and the author need to give explanation for the results. there are another minor correction required to improve the quality of the article.

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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: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (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: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: No

Reviewer #3: Yes

Reviewer #4: 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: Yes

Reviewer #3: Yes

Reviewer #4: 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: Yes

Reviewer #3: Yes

Reviewer #4: 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: The manuscript titled "Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon" was well written and contains good information. However, Demographic, socioeconomic and nutritional variables have a relationship with each other and affect each other, and they should be included in the multivariate statistical analysis together.

And it was necessary to determine the basis on which the variable is included for the multivariate examination.

In addition, the authors treat the Odd ratio test on the basis that it quantites the association between the variables and this is incorrect. Odd ratio quantifies the strength of the association between two events. There are other tests to do this, such as a risk ratio (RR). On the other hand, we cannot say that the risk increases by 2.4 (table 2 etc..), in fact, the increase is only 1.4 because one is not calculated, as it means that there is no difference.

Other minor corrections include:

It would be better for the author to indicate the average temperatures in the study area. For the phrase "Toxoplama infection" throughout the manuscript, the the genus Toxoplasma should be written in Italic. The age group 40 and more should be corrected (Table 1). Use three decimal digits for P value results.

Reviewer #3: Lines 67-69: Authors to rephrase the paragraph and provide references regarding the seroprevalence and risk factors associated with T. gondii infection among individuals with human immunodeficiency virus (HIV) from different areas of the world.

Lines 326-327: please delete BMC, the journal is Parasite Vectors

Reviewer #4: please check the uploaded file for corrections.

For the discussion need to find researches to support your findings

**********

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 #3: No

Reviewer #4: 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.

Submitted filename: PONE-D-20-04369_R1_reviewer.pdf

Click here for additional data file.

10 Jun 2021

Response to Reviewers.

Dear reviewers’ kindly fine in this later the response to the questions posted.

The average annual temperature of the study area is 19.30c. During the risk factor analysis of the data, the demographic, socioeconomic and nutritional characteristics were all included together in the multivariate analysis. It was done after a bivariate analysis were performed.

The investigator captured the aspect of clinical manifestation by using a questionnaire tool. These questions were asked to the participants to consult them of the history of clinical manifestation event related to that of the illness.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

13 Jul 2021

PONE-D-20-04369R2

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

PLOS ONE

Dear Dr. Enah,

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.

There are few grammatical/typo errors that need to be addressed before proceed to the next step as suggested by the reviewers.

Please submit your revised manuscript by Aug 27 2021 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:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohammed Abdelfatah Mosa Alhoot, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Comments to authors:

The manuscript was improved. However, minor edits should be made.

Lines 69-71: please delete “It would be useful to present data regarding the seroprevalence and risk factors associated with the infection amongst human immunodeficiency virus (HIV) of T. gondii from different areas of the world.”

I suggest to replace with this statement:

“Knowledge of T. gondii seroprevalence and its associated risk factors is important to predict the risk of infection in humans.”

Reviewer #4: The authors have adequately addressed comments raised in previous review. This time only 2 corrections in text (typing errors)

**********

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 #3: No

Reviewer #4: 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.

Submitted filename: PONE-D-20-04369_R2_reviewer.pdf

Click here for additional data file.

14 Aug 2021

Responses to Review Comments for Manuscript: PONE-D-20-04369R2

Thank you for pointing out the corrections that should be done on our manuscript. We have dealt with them as recommended/advised; all the points raised have been addressed; grammatical and typographical errors inclusive. We have equally removed reference 24; this is because it has not been cited anywhere in the manuscript. We have also highlighted the changes introduced. We hope we have answered all the questions and concerns satisfactorily and therefore look forward to your anticipated action.

REVIEWER 3

Comment: Please delete ‘’ It would be useful to present data regarding the seroprevalence and risk factors associated with the infection amongst human immunodeficiency virus (HIV) of T. gondii from different areas of the world.

Response: The statement has been deleted and replaced with‘’Knowledge of T. gondii seroprevalence and its associated risk factors is important to predict the risk of infection in humans’’ as suggested. Please, see lines 71 – 72 on the revised manuscript with tract changes

Raymond B. NYASSA, PhD

Corresponding author

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

20 Aug 2021

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

PONE-D-20-04369R3

Dear Dr. Enah,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Mohammed Abdelfatah Mosa Alhoot, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

23 Nov 2021

PONE-D-20-04369R3

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

Dear Dr. Nyasa:

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

Dr. Mohammed Abdelfatah Mosa Alhoot

Academic Editor

PLOS ONE