Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model.

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n = 10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0-15, (b) acute: 16-30, (c) sub-acute: 31-45 and (d) chronic: 46-60 dpi. Other virulence biomarkers identified included: pre-patent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B), pentamidine, diminazene aceturate and suramin, using mice groups (n = 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. A cure rate of at least 80% was achieved for all test isolates with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr clones from a single HAT focus and confirms that this variations is not a significant determinant of isolate sensitivity to anti-trypanosomal drugs.

Introduction

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne parasitic disease. It is caused by infection of humans with protozoan parasites belonging to the genus Trypanosoma. HAT is caused by two subspecies of trypanosomes, namely Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense [1]. They are transmitted to humans by tsetse flies (Glossina genus) [2]. Trypanosoma brucei gambiense is found in countries in West and Central Africa and causes a chronic infection [3]. A person can be infected for months or even years without major signs or symptoms of the disease [4]. When more evident symptoms emerge, the patient is often already in an advanced disease stage where the central nervous system is affected. Trypanosoma brucei rhodesiense is found in countries in eastern and southern Africa and causes an acute infection. Symptoms manifest within 2–4 weeks of an infective tsetse fly bite [3]. HAT develops in two stages namely, the early (hemolymphatic) and the late (meningo-encephalitic) stage. In the early stage of the disease, parasites proliferate in the blood and lymphatic system while in the late stage, parasites penetrate the blood brain barrier (BBB) and persist and proliferate in the central nervous system (CNS), causing an encephalitic reaction that leads to death if untreated or inadequately treated [5]. For first stage infections, there are no specific clinical signs and symptoms in both forms of the disease; fever, headache and loss of appetite are common [1] as well as anaemia in the monkey model [6]. With T.b. rhodesiense infections, first signs and symptoms are observed a few weeks after infection [1]. However, a mild form of chronic T. b. rhodesiense infections with incubation times of several months has been reported in Zambia [7]. The acute and the chronic HAT infections caused by T. b. rhodesiense in different foci differ in both their inflammatory response and pathology. According to Maclean et al (2008), the pathology encountered in the acute HAT infections is characterized by elevated Tumor necrosis factor alpha (TNF-α) while that encountered in the chronic HAT infections is characterized by elevated transforming growth factor (TGF-β) [8].

Treatment of Trypanosoma brucei rhodesiense infections involves the use of early stage drugs such as pentamidine and suramin [9] and late stage drugs such as melarsoprol; melarsoprol is the only drug recommended by WHO for treatment of late-stage T b rhodesiense infection, but can be lethal to 5% of patients owing to post-treatment reactive encephalopathy [10]. HAT therapy is further complicated by reports of drug resistance in different foci, including against suramin and melarsoprol in Tanzania [11] and against melarsoprol in south Sudan [12]. In their study, Pyana and colleagues [13] suggested that investigations into treatment failure in HAT and use of alternative drugs or treatment regimens should not only focus on differential genotypes of the parasites but also on differential virulence and tissue tropism as possible causes. The present study was therefore designed to investigate the occurrence of differential virulence of isolates recovered from Western Kenya/ Eastern Uganda HAT focus and the potential role of this variations on isolate sensitivity to anti-trypanosomal drugs using the mouse model. Studies on disease pathogenesis, parasite virulence, drug sensitivity and identification of new potential drug targets and staging biomarkers are commonly carried out in the mouse model based on its cost effectiveness, genetic similarity to humans estimated to be 85%, and ethical limitations of carrying out such studies in higher animal models or humans [14-16]. The study will also avail well characterized T.b.rhodesiense isolates for future studies.

Materials and methods

Ethics

All experimental protocols and procedures used in this study involving laboratory animals were reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of Kenya Agricultural and Livestock Research Institute–Biotechnology Research Institute (KALRO-BioRI) Ref: C/Biori/4/325/II/53)

Experimental animals

The study used 6to 8 weeks old male Swiss White mice, each weighing 25–30 g live body weight. The animals were obtained from the Animal Breeding Unit at KALRO-BioRI, Muguga. The mice were housed in standard mouse cages and maintained on a diet consisting of commercial pellets (Unga® Kenya Ltd). All experiments were performed according to the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of KALRO-BioRI. Briefly, water was provided ad libitum. All mice were acclimatized for two weeks, during which time they were screened and treated for ecto and endoparasites using ivermectin (Ivermectin®, Anupco, Suffolk, England). During the two-week acclimation period, pre-infection data were collected on body weights and packed cell volume once a week prior to parasite inoculation.

Trypanosomes and cloning

Twelve T.b. rhodesiense trypanosome stabilates (KETRI 2482, 2487, 3304, 3305, 3380, 3664, 3798, 3800, 3801, 3803, 3926, 3928) were selected from the KALRO-BioRI specimen bank. Cloning was carried out as described by [17]. Briefly, the trypanosome stabilates were inoculated into mice immunosuppressed using cyclophosphamide at 100 mg/kg for three consecutive days (total dose 300mg/kg) body weight (bwt) as previously described [18]. Animals were monitored daily for parasitaemia. When the mice attained a parasitaemia score of 3.2.x107 trypanosomes/mL [19], they were bled from the tail vein and the blood sample appropriately diluted using a mixture of PSG pH 8.0 and guinea pig serum in the ratio of 1:1. Using the hanging drop method [20], a single trypanosome was then picked using a syringe with a 25 gauge needle suspended in at least 0.2mls PSG pH 8.0 and injected intraperitoneally (ip) into a single immunosuppressed mouse. This was replicated ten times to increase the chances of success. Infected mice were then monitored for parasitaemia daily [19]. Any of the ten mice which became parasitaemic was euthanized using concentrated carbon dioxide, bled from the heart and the harvested trypanosomes cryopreserved in PSG pH 8.0 in 20% glycerol as a clone stabilate.

Virulence studies

Design of virulence study

Male Swiss White mice were housed in groups of 10 in standard mouse cages containing wood shavings as bedding material. The cryopreserved cloned parasites were thawed, and injected ip into immunosuppressed donor Swiss White mice for multiplication. The mice were euthanized using carbon dioxide [21] at peak parasitaemia and blood collected from the heart in EDTA for quantification as previously described [22]. The ten mice in each cage were infected with one Tbr clone, with each mouse receiving 1x104 trypanosomes injected intraperitoneally. The infected mice were monitored for pre-patent period, parasitaemia progression, PCV, body weight and survival time as virulence biomarkers. A control group of 10 non-infected mice was included in the study and monitored for changes in PCV, body weight and survival times.

Pre-patent period and parasitaemia progression

Blood for estimation of parasitaemia levels was collected daily for the first 14 days and thereafter three times in a week from each mouse using the tail tip amputation method [23]. The PP and parasitaemia progression were determined using the rapid matching method of [19, 24]. The infected mice were monitored for 60 days post infection.

Packed cell volume (PCV) and body weight changes

PCV was determined as outlined by [25]. Body weight (bwt) was measured once a week using a (Mettler Tolendo PB 302 ®, Switzerland) digital balance [22].

Survival times and virulence classification

The classification of trypanosome virulence was based on the survival of the infected mice as previously described [26]. The twelve T.b. rhodesiense clones were placed into four classes of virulence based on the survival of 60% or more of the infected mice as follows: very-acute (0–15 days), acute (16–30), sub-acute (31–45) and chronic classes (46–60). Each mouse’s survival time was determined on the basis of a ≥ 25% decline in PCV and consistently high parasitaemia levels of 1x109/ml for at least two consecutive days as previously described [27]. The mice were immediately euthanized by CO2 asphyxiation following the Institutional Animal Care and Use Committee (IUCAC) guidelines as earlier described by [28] and recorded as dead animal. Mice surviving at 60 dpi were equally euthanized, survival time recorded as 60 days and categorized as censored data.

Drug sensitivity study

Initially, sensitivity patterns for KALRO-BiORI laboratory reference isolates considered drug resistant (KETRI 2538) or drug-sensitive (KETRI 3738) were determined for Melarsoprol (Arsobal®, Aventis), Diminazene aceturate [(Veriben®, Ceva, France), Pentamidine (Pentacarinat®-Sanofi, UK) and Suramin (Germanin® Bayer), using dose rates ranging from 1–40 mg/kg body weight (Table 2) in order to identify cut-off points for characterizing isolates as drug resistant. Thereafter, the T. b. rhodesiense test clones were evaluated for sensitivity to the same drugs (Table 3). An isolate was considered drug-resistant if 2/5 (40%) of the infected and treated mice relapsed [11] after having been treated at 20mg/kg bwt.

Table 2

Results of drug sensitivity evaluation of reference KALRO-BioRI sensitive and resistant T b rhodesiense isolates.

Sensitive Isolate KETRI 2537				Resistant isolate KETRI 2538
Drug	Drug dose (mg/Kg)	Mice cured/5	Status	Drug dose (mg/Kg)	Mice cured/5	Status
MelB	40	5	(s)	40	5	(S)
	20	4	(s)	20	0	(R)
	10	5	(s)	10	0	(R)
	5	5	(s)	5	0	(R)
	2.5	5	(s)	2.5	0	(R)
	1	0/5	(R	1	0	(R)
diminazene aceturate	40	5	(s)	40	5	(S)
	20	5	(s)	20	0	(R)
	10	4	(s)	10	0	(R)
	5	5	(s)	5	0	(R)
	2.5	5	(s)	2.5	0	(R)
	1	2	(R)	1	0	(R)
Pentamindine	40	5	(s)	40	5	(S)
	20	5	(s)	20	1	(R)
	10	5	(s)	10	1	(R)
	5	4	(s)	5	0	(R)
	2.5	2	(R)	2.5	0	(R)
	1	0	(R)	1	0	(R)
Suramin Control	40	5	(S)	40	5	(S)
	20	5	(S)	20	5	(S)
	10	5	(s)	10	5	(S)
	5	5	(s)	5	5	(S)
	2.5	4	(s)	2.5	2	(R)
	1-	110	(R)	1-	010	(R)

Key:- Not treated; The mice groups (n = 5) were treated 24hours post inoculation with the isolates and monitored for 60 days post treatment. An isolate is coded as sensitive (S) when at least 4/5 mice survived for at least 60 days without trypanosome relapse. All other results are coded as resistant (R).

Table 3

Results of drug sensitivity evaluation of T b rhodesiense clones in the mouse model.

Stab. No KETRI	Virulence Class	Pentamidine Mice cured/ total treated		Melarsoprol Mice cured/ total treated		diminazene aceturate Mice cured/ total treated		Suramin Mice cured/ total treated
	Very-acute	5mg/kg	20mg/kg	1mg/kg	20mg/kg	2.5mg/kg	20mg/kg	2.5mg/kg
2482		5/5	5/5	5/5	5/5	4/5 (1)	5/5	2/2 (3)b
3304		5/5	5/5	5/5	5/5	5/5	5/5	5/5
3803		4/4 (1)b	5/5	5/5	4/4(1)b	5/5	5/5	2/2(3)b
2487	Acute	4/5 (1)a	5/5	5/5	5/5	5/5	5/5	5/5
3801		5/5	5/5	5/5	5/5	5/5	5/5	5/5
3798	Sub- acute	5/5	5/5	5/5	5/5	5/5	5/5	5/5
3926		4/5(1)a	5/5	5/5	5/5	5/5	5/5	3/3(2)b
3380	Chronic	5/5	5/5	5/5	5/5	5/5	5/5	5/5
3928		4/4 (1)b	4/4 (1)b	4/4 (1)b	5/5	1/1(4)b	5/5	5/5

Key: The mice were treated with single doses of various anti-trypanosomal drugs at 24 hours post infection and monitored for 60 days post treatment; a, number of mice which relapsed in each group during the 60 days of post-treatment monitoring; b, number of mice which died without parasitaemia relapse. All the isolates recorded at least 80% cure rates to all drug dose regimens and were therefore classified as sensitive.

Suramin and Pentamidine drugs (100% w/w) for the highest dosage of 40mg/kg bw were prepared by dissolving 40mg of these drugs in 10mls distilled water to give a concentration of 4 mg/ml. Diminazene aceturate (44.44% w/w active ingredient) for the highest dosage of 40mg/kg bw was prepared by dissolving 90mg of the drug powder in 10 mLs distilled water to give a concentration of 4mg/ml, whereas Melarsoprol (5 Ml vials of 180 mg) was first prepared by mixing (vortex) 1 Ml of the stock solution to 4 Ml of 50% propylene glycol to give a concentration of 7.2mg/ml (72mg/kg). This was further diluted to 40mg/kg by mixing(vortex) 5.6 ml of the 7.2mg/ml with 4.4 Mls of 50% propylene glycol to give a concentration of 4mg/ml (40mg/kg) The drug solutions for the 40mg/kg dose of each drug were then diluted serially using distilled water to give dosages for the 20, 10, 5, 2.5, 2, 1mg/kg.

Statistical analysis

Analysis was carried out to test if there are significant differences between the four classes of virulence using PP, parasitaemia progression, PCV, body weight changes and survival as the response variables. The data obtained from the study were summarized using descriptive statistics. The general linear model in SAS was used to test significance at p<0.05 level, between the means of the four virulence classes. Survival data analysis was carried out employing the Kaplan–Meier method on StatView (SAS Institute, Version 5.0.1) statistical package for determination of survival distribution function. Rank tests of homogeneity were used to determine the effect of virulence on host survival time [29].

Results

Survival time and classification

All control mice survived to the end of the experimental period of 60 days and their data (mean survival period) were therefore categorized as censored. The 12 T. b. rhodesiense clones exhibited variation in survival time and were classified into four classes of virulence based on these survival time data as shown (Table 1). A total of 3/12 clones were very acute, 3/12 were acute, 2/12 sub-acute and 4/12 chronic (Table 1).

Table 1

Changes in virulence biomarkers in mice infected with twelve clones of Trypanosoma brucei rhodesiense.

Class	Clone ID	Locality of isolation	Iso. yr	PP passage No	Peak Para.	DPP	MST
very-acute	KETRI 2482	Lumino, Uganda	1969	5±0 7	1x106	8±0.2	9±0.4
	KETRI 3304	Lugala, Uganda	1971	5±0 64	7.6x108	7.8±0.4	9±0.4
	KETRI 3803	Busia, Kenya	1961	4±0 2	9.3x108	6.4±0.2	8.2±0.3
	Group mean ±SE			4.7±0.9	8.9x108	7.4±0.5	8.8±0.2
Acute	KETRI 2487	Busoga, Uganda	1972	4±0 5	6.2x108	7±0	18.1±0.7
	KETRI 3800	Busia, Kenya	2000	5.2±0.1 2	1.2x108	6±0	26.0±2.0
	KETRI 3801	Busia, Kenya	1989	6±0 1	6.8x107	7.3±0.3	20.4±0.8
	Group mean ±SE			5.03±0.16	1.7x108	6.7±1.3	21.6±1.0
Sub-acute	KETRI 3798	Busia, Kenya	1989	5.3±0.16 2	1.6x108	9.3±1.7	28.2±2.0
	KETRI 3926	Busoga, Uganda	1972	5±0 8	1.7x108	6.6±0.2	38.9±1.6
	Group mean ±SE			5.2±0.8	1.5x108	7.9±0.9	33.6±1.8
Chronic	KETRI 3928	Tororo, Uganda	1992	6.3±0.3 2	7.9x107	7.0±0	51.8±4.1
	KETRI 3664	Busia, Kenya	1997	6.0±0 2	4.8x107	7.2±0.3	45.6±1.6
	KETRI 3380	Busoga, Uganda	2000	5.9±0.5 3	1.3x108	7.3±0.2	55.5±2.3
	KETRI 3305	Lugala, Uganda	1971	6.7±0.2 5	3.2x108	8.7±0.3	46.7±5.1
	Group mean ±SE			6.3±0.2	1.1x108	7.5±0.2	49.9±1.8

Control Non-infected          -          -          -          -          >60

Key: PP-pre-patent period, Iso Yr–year of isolation, Par-parasitaemia, DPP -days to peak parasitaemia, MST-mean survival times,—No data

The mean survival time (MST) of isolates categorized as very acute ranged from a mean ± SEM of 8.7±0.2 days (KETRI 2482) to 9.0 ± 0.4 for KETRI 3304 (Table 1, S1 Fig (i)). These survival time data were not statistically different from each other (p > 0.05). Mice infected with the acute clones had MST ranging from a mean±SEM of 18.1±0.7 (KETRI 2487) to 26 ±2.0 for T.b rhodesiense clone KETRI 3800 (S1 Fig (ii)); these data were significantly (p < 0.0001) different from each other (S1 Fig (ii)). In mice infected with T. b. rhodesiense clones characterized as sub-acute, the survival time ranged from a mean ± SEM of 28.2±2.0 for KETRI 3798 to mean±SEM of 38.9 ± 1.6 for KETRI 3926 (S1 Fig (iii)) and were significantly (p<0.0001) different from each other. Mice infected with the T. b. rhodesiense clones characterized as chronic had MST values ranging from a mean±SEM of 45.6±1.6 for KETRI 3664 to a mean ± SEM of 55.5 ± 2.3 days for KETRI 3380 (S1 Fig (iv)) and were significantly (p<0.001) different from each other.

The survival time data for all the isolates in each virulence class were grouped together for the purpose of comparison among the groups. The overall MST was 8.7±0.2 for the very acute clones was, 21.6±1.0 for the acute clones 33.6±1.4 for the subacute clones and 50.0±1.8 for the chronic clones (Table 1, Fig 1). The Wilcoxon and Logrank tests had a p-value of 0.001, each showing that the MST of isolates in the different classes were significantly different.

Fig 1

The survival times for mice (n = 10) infected with twelve T. b. rhodesiense clones.

The clones were classified as (i) very-acute (0–15 dpi), (ii) acute (16–30 dpi), (iii) sub-acute (31–45 dpi), (iv) and chronic Tbr (46–60 dpi) all classes of virulence grouped together.

Pre-patent period and parasitaemia progression

The mean ±SEM pre-patent period in mice infected with very acute isolates ranged between 4±0.0 (KETRI 3803) to 5±0.0 (KETRI 2482 and 3304) and 4.7±0.09 when isolates are considered as a group (Table 1). The mean pre-patent period in mice infected with the acute class of the isolates ranged between 4±0.0 (KETRI 2487) and 6.0±0.0 (KETRI 3801) and 5.03±0.2 when they are considered as a class. With mice infected with sub-acute clones, the mean pre-patent period ranged between 5.0±0.0 (KETRI 3926) and 5.3±0.2 (KETRI 3798) and 5.2±0.08 when considered as a class. The pre-patent period for the mice infected with the chronic isolates was in the range of 5.9±0.5 (KETRI 3380) to 6.7±0.2 (KETRI 3305) and 6.3±0.2 dpi when considered as a class (Table 1). When the pp data were grouped together for isolates in each virulence class, the mean ±SEM were 4.7±0.09 (very acute class), 5.03±0.2 (acute class), 5.2±0.08 (sub-acute class and 6.3±0.2 (chronic class). Despite the apparently increasing trend of these data, these differences were not statistically significant (p>0.05).

The parasitaemia patterns, results of the mean peak parasitaemia (±SE), and time (days) to peak parasitaemia (DPP) of the T. b. rhodesiense clones as shown in (Table 1, Fig 2). In mice infected with the three (3) very-acute clones, the mean parasitaemia of each clone was characterized by a single wave as compared to two waves for mice infected with the acute, subacute and chronic clones (S2 Fig (i); S2 Fig (ii), S2 Fig (iii) and S2 Fig (iv)).

Fig 2

Parasitaemia progression in mice (n = 10) infected with the four classes of T. b. rhodesiense clones.

(i) Very-acute, (ii) acute, (iii) sub-acute and (iv) chronic clones all classes of virulence grouped together.

Trypanosoma b. rhodesiense clone specific variation was observed in the mean peak parasitaemia and time (days) to peak parasitaemia (Table 1). In the very acute virulence class, clone KETRI 3803 attained the highest mean peak parasitamia of 9.3 x 108 trypanosmes/mL of blood as compared to 1 x 106 trypanosomes/mL of blood for clone KETRI 2482 (Table 1), a 100-fold difference; these differences were statistically significant (p < 0.001). The KETRI 3803 clone attained peak parasitaemia faster (6 days) as compared to 8 days for the KETRI 2482 (Table 1). The third clone in this class, KETRI3304 was intermediate with respect to both parameters. Out of the three clones classified as acute, KETRI 3800 and 3801 had significantly (p 0.001) lower mean peak parasitamia parasitaemia when compared to KETRI 2487 (S2 Fig (ii)). A comparable pattern was observed for the sub-acute clones, with KETRI 3926 showing a significantly (p<0.001) lower parasitaemia than clone KETRI 3798 (S2 Fig (iii)). Similarly, clone KETRI 3305 recorded significantly lower parasitaemia (S2 Fig (iv)) when compared to the rest of the Tbr clones classified as chronic.

When parasiataemia data for T. b. rhodesiense clones in each virulence class were grouped together and compared, parasitaemia was significantly (p<0.001) higher in mice infected with very- acute clones (Fig 2).

Packed cell volume (PCV)

The pre-infection PCV data for all infected and control mice groups (Fig 3) were not statistically different (p > 0.05). The PCV of the infected mice declined significantly (p < 0.001) following infection when compared with the PCV of the non-infected control mice which remained largely constant throughout the duration of the study (Fig 3). However, the onset and severity of the anemia, as shown by the decline in PCV, was most prominent for mice infected with the isolates classified as very acute (Fig 3). In these mice, the PCV declined significantly (p<0.001), from 49.7±0.8 at baseline (day 0) to 26.0±0.5 at 14 dpi equivalent to 47.7% decline. The lowest infection-related decline in PCV (Fig 3) was recorded in the mice infected with isolates classified as chronic clones, with the PCV declining from 49.6±0.9at baseline to 43.5±1.0at 14 dpi (12.3%).

Fig 3

Mean ± SE PCV decline in mice (n = 10) infected with T.b. rhodesiense (i) very-acute isolates, (ii) acute isolates, (iii) sub-acute isolates and (iv) chronic isolates clones all classes of virulence grouped together.

The changes in PCV varied significantly between T. b. rhodesiense clones of the same virulence class (Fig 3). In mice infected with very-acute clones, KETRI 3304 infected mice recorded the highest decline from 48.25±1.6 at day 0 to 39± 2.4 at 7 dpi equivalent to 19.2% (S3 Fig (i)) whereas within the acute clones infected mice, KETRI 2487 infected mice recorded the highest decline from 47.7±1.8 at day 0 to 38.8±1.4 at 7 dpi equivalent to 18.7% (S3 Fig (ii)). In mice infected with the sub-acute clones, KETRI 3978 recorded the highest decline from 53.4±1.1 at day 0 to 47.9±0.6 at 7dpi equivalent to 10.3% (S3 Fig (iii)) and in mice infected with the chronic clones, KETRI 3305 infected mice registered the highest decline from 54±2 at day 0 to 47±1.4 at 7dpi equivalent to 13% (S3 Fig (iv)).

When the mean PCV data of all isolates in each virulence class were grouped together and compared, the decline in PCV was significantly (p<0.001) higher in mice infected with very-acute clones (Fig 3); the PCV of mice infected with the very acute clones declined from 49.7±0.8 to 26.0±0.5 at 14 DPI by an average of 47.7% as compared to an average decline of 12.3% for mice infected with the chronic Tbr clones.

Body weight

The mean ±SE pre-infection body weight data for infected and control groups (Fig 4) were not statistically different (p > 0.05). Between 7 and 14 days post infection, all infected mice groups exhibited a decline in mean body weight (S4 Fig (i)–S4 Fig (iv)) while the body weight of un-infected control mice did not change (Fig 4). However, mice groups infected with clones classified as acute, sub-acute and chronic exhibited recovery of their body weights starting 14 dpi. Mice group infected with isolates in the very acute virulence class did not survive beyond 14 dpi (Fig 4).

Fig 4

Mean ± SE body weight changes in mice (n = 10) infected with T.b. rhodesiense.

(i) very-acute isolates, (ii) acute isolates, (iii) sub-acute isolates and (iv) chronic isolates all classes of virulence grouped together.

Drug sensitivity results

The results of the drug-sensitivity testing for the reference sensitive (Tbr KETRI 3738) and drug-resistant (Tbr KETRI 2538) isolates are shown in (Table 2). The reference drug-resistant isolate was confirmed to be resistant to melarsoprol, Pentamidine and Diminazene aceturate at dose rates ranging from 1–20 mg/kg body weight (Table 2). However, infected mice were cured with all three drugs at a dose rate of 40 mg/kg body weight. With respect to suramin, the reference resistant isolate was sensitive to all doses equal to or greater than 5 mg/kg body and is therefore characterized as sensitive (Table 2). On the other hand, reference drug-sensitive isolate was confirmed to be sensitive to all doses of melarsoprol, ranging from 1–40 mg/kg bwt. It was also fully sensitive to diminazene aceturate at dose rates ranging from 2.5-40mg/kg bwt. The reference sensitive isolate was sensitive to pentamidne at all doses above 4 mg/kg bwt (Table 2). It was also sensitive to all doses of suramin equal to or greater than 2.5 mg/kg (Table 2)

The results of drug sensitivity experiments for the test Tbr clones are summarized in (Table 3). All the isolates recorded at least 80% cure rates for all drug dose regimens evaluated in this study (Table 3) and were therefore classified as sensitive. However, a few cases of relapsewere observed in 1/5 (20%) mice infected with KETRI 2482 (very-acute group) and treated with diminazene aceturate at 2.5mg/kg, and KETRI 2487 (acute) and KETRI 3926 (sub-acute) treated with pentamidine at 5mg/kg. In mice infected with KETRI 3928 (Chronic), 4/5 treated with diminazene aceturate at 2.5mg/kg and 5/5 treated at 20 mg/kg died at 47 days post treatment and without detectable parasites (Table 3), This is presumably due to pneumonia resulting from cold environment caused by a leaking water bottle. No relapses were observed in mice groups that were treated with either melarsoprol (1 and 20 mg/kg) or suramin at 2.5 mg/kg (Table 2)

Discussion

In this study, we characterized the virulence and anti-trypanosomal drug sensitivity patterns of 12 T. b. rhodesiense cloned stabilates. We used T. b rhodesiense clones because they represent a homogeneous population of genetically identical trypanosomes thus making subsequent studies based on these clones more reproducible and reliable as previously reported [30]. The results demonstrated the existence of variation in virulence of T. b. rhodesiense cloned stabilates (Table 1) which is interesting because all study isolates were originally recovered from western Kenya and eastern Uganda, regions that are considered to belong to the same Busoga focus of HAT. Our results are in agreement with a study on a number of isolates from eastern Uganda in mice which showed that distinct acute and chronic strains of T. b. rhodesiense circulated in the focus [31]. The results are also also in agreement with a previou report of variations in virulence of T. b. gambiense isolates [32].

We used mean survival time (MST) of mice post-infection as the main indicator of virulence as previously reported [26, 27, 33]; and observed that the Tbr isolates were well distributed among the four virulence classes. Infective isolates that allowed mice to have long survival times, hence chronic infections, may indicate presence of enriched population of stumpy forms which aids in prolonging host survival and enhancing the probability of parasite transmission [34]. The mean survival times for the very acute clones was 8.7 days suggesting the hosts were overwhelmed by the first parasitaemia peak before the proliferating slender forms differentiated into short stumpy forms [35]. The majority (11/12) of the Tbr clones used in this study had undergone a minimal number of (1–8) passages since isolation (Table 2). The only isolate that had undergone a significant (64) number of passages (Table 2) did exhibit notably different virulence characteristics from the other clones in the class, suggesting therefore that the observed differences in isolate virulence are an intrinsic attribute as previously reported [36].

Parasitaemia progression differed significantly among Tbr isolates assigned to different virulence classes on the basis of survival time. This finding is in agreement with previous studies in which virulence of different species of trypanosomes was characterised using parasitaemia, intensity of anaemia (PCV) and weight loss experienced by the host during the infection period [27]. In our study, parasitaemia of isolates in the very-acute virulence class was represented by a single wave whereas the acute, sub-acute and chronic virulence classes were represented by two waves. (Fig 2). This is in agreement with studies by [37] who observed that acute infections result from uncontrolled proliferation of the slender trypanosome forms without differentiation into short stumpy forms and hence kills the host before tsetse transmission takes place [37]. In contrast, chronic infection is characterized by appearance of progressive waves of parasitaemia, with each distinct wave being composed of trypanosomes with antigenically distinct coats, and with parasites easily differentiating into the transmissible short stumpy forms. This perhaps explains why highly virulent trypanosomes are not easily transmissible as was observed by [22] that tsetse flies infected with chronic T. b. brucei recorded highest mature infection as opposed to those infected with highly virulent trypanosomes. Our results are important as they reveal that the majority of T. b. rhodesiense infections are in the bracket of (acute, sub-acute and chronic) classes of virulence and can easily be transmittable.

In the present study, all infected mice recorded a decline in PCV signifying the development of T. b. rhodesiense induced anemia. Our observation was in agreement with previous studies which reported anaemia as a key feature both in humans [38] and in the monkey model [6]. As with parasitaemia and survival time parameters, the development of anemia was significantly pronounced in mice infected with very-acute clones. This finding is consistent with observations by [39] who reported that acute infection of mice with Trypanosoma cruzi was characterized by an exponential growth of parasites and high mortality accompanied by anemia. A similar observation was made by [27] in mice infected with Trypanosoma evansi. In contrast, anaemia in mice infected with clones in the other various classes of virulence (acute, sub-acute and chronic) stabilized or recovered characteristic of the chronic phase anaemia [40]. The severity of anemia is determined by parasite virulence, time lag from infection to therapeutic intervention and individual host differences [41].

Our results showed a decline in body weight in the early days of infection (7–14 dpi)

This decline was however not significant. Our observation is important as it confirms a previous observation [22] that body weight alone cannot conclusively serve as a virulence biomarker. Previous authors [42] attributed decline in body weight to reduced food intake. In our study, we did not measure the food intake. The failure by infected mice to register a significant decline in body weight calls for further investigation on the causes of body weight changes in animals infected with trypanosomes especially after previous studies have recorded an increase in body weight in T.evansi [27] and in T. b. brucei or T. congolense [22] infected mice.

Our results on drug sensitivity tests showed that all the study isolates were sensitive to melarsoprol, pentamidine, diminazene aceturate and suramin. The sensitivity of these isolates to suramin and melarsoprol is significant since currently they are the only drugs of choice recommended by WHO (2018) to treat early and late stages of Tbr HAT respectively. On the other hand the sensitivity of the Tbr isolates to diminazene aceturate, is an indicator of the utility of these drug when administered to livestock reservoirs of Tbr isolates as practiced in disease HAT control programmes in endemic countries [43] Interestingly, however, the single cases of relapses encountered in mice infected with KETRI 2482 (very- acute virulence class), KETRI 2487 (acute virulence class) and 3926 (sub-acute virulence class) were all against the two diamidines (pentamidine or dimainazene) but not against suramin or melarsoprol (Table 3) which is consistent with clinical practice of not using these specific diamidines to treat Tbr HAT (WHO, 2018). Overall, the fact that the test isolates were all sensitive (at least 80% cure rates) to the drugs suggests there was no relationship between isolates’ virulence and their sensitivity to anti-trypanosomal drugs which is in agreement with previous studies [44, 45]. In a study by Sokolova et al [46], these authors observed that resistance to nifurtimox did not compromise parasite virulence. This is despite previous studies reporting that drug resistant trypanosome have reduced virulence [47, 48].

The KALRO-BioRI reference isolate considered to be drug resistant was confirmed in this study to be resistant to melarsoprol, pentamidine and diminazene aceturate (Table 2). In general drug resistance is attributed to reduced drug uptake due the mutation or absence of a drug uptake gene [49] as well as by enhanced drug export, mediated by a multidrug resistance-associated protein [50]. The uptake of the three drugs, melarsoprol, pentamidine and diminazene is mediated by the P2 transporter [12, 51, 52] which explains why resistance to all three drugs is linked. In contrast, uptake of suramin by trypanosomes is not mediated by the P2 transporter, hence the reason why the trypanosome, KETRI 2538, retains sensitivity to suramin

In summary, this study has found that there is variation in virulence of cloned stabilates made from field isolates recovered from western Kenya/eastern Uganda HAT focus. Virulence is attributed to the production by the blood stream forms of membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vehicles (EVs). This (EVs) contain several flagellar proteins that contribute to virulence [53]. Since our study was based on laboratory cloned T. b. rhodesiense stabilates, however, future studies should utilize the parent primary isolates. Our results are important as they have demonstrated that virulence is not a hindrance in the control of trypanosomiasis by chemotherapy.

(i): Mean survival times in mice infected with the very-acute clones of Trypanosoma brucei rhodesiense. (ii): Mean survival times in mice infected with the acute clones of Trypanosoma brucei rhodesiense. (iii): Mean survival times in mice infected with the sub-acute clones of Trypanosoma brucei rhodesiense. (iv): Mean survival times in mice infected with the chronic clones of Trypanosoma brucei rhodesiense.

(DOCX)

Click here for additional data file.

(i): Mean parasitaemia progression in mice infected with the very-acute clones of Trypanosoma brucei rhodesiense. (ii): Mean parasitaemia progression in mice infected with the acute clones of Trypanosoma brucei rhodesiense. (iii): Mean parasitaemia progression in mice infected with the sub-acute clones of Trypanosoma brucei rhodesiense. (iv): Mean parasitaemia progression in mice infected with the chronic clones of Trypanosoma brucei rhodesiense

(DOCX)

Click here for additional data file.

(i): Mean PCV decline in mice infected with the very acute clones of Trypanosoma brucei rhodesiense. (ii): Mean PCV decline in mice infected with the acute clones of Trypanosoma brucei rhodesiense. (iii): Mean PCV decline in mice infected with the sub-acute clones of Trypanosoma brucei rhodesiense. (iv): Mean PCV decline in mice infected with the chronic clones of Trypanosoma brucei rhodesiense.

(DOCX)

Click here for additional data file.

(i): Mean body weight changes in mice infected with the very acute clones of Trypanosoma brucei rhodesiense. (ii): Mean body weight changes in mice infected with the acute clones of Trypanosoma brucei rhodesiense. (iii): Mean body weight changes in mice infected with the sub-acute clones of Trypanosoma brucei rhodesiense. (iv): Mean body weight changes in mice infected with the very chronic clones of Trypanosoma brucei rhodesiense.

(DOCX)

Click here for additional data file.

26 Feb 2020

PONE-D-20-02583

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

PLOS ONE

Dear Mr. Ndungu,

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I) Why was the control group (not infected mice) not included in Table 1. Although this group is not mentioned in Materials and Methods, it is mentioned in results in lines 200 and 213. This would add more credence to the results.

ii) In line 188, please mention the groups that the mice infected with very acute clone were being compared to.

iii) Line 52, add a full stop after "infection" and "an" before "infection"

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https://doi.org/10.1371/journal.pntd.0001857

https://doi.org/10.1017/S0031182017002359

https://www.afro.who.int/health-topics/trypanosomiasis-african

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Reviewer #1: This is an interesting comparative paper in which 12 clones are compared. These clones all originate from stabilates that were originally collected in the same HAT area, which adds particular value to the paper. However, there are some issues that dampen the value of the paper as it lacks scientific data that could be very valuable for others. There is also one scientific concept that according to this reviewer should be taken into account for the discussion section.

Main problem:

Figures 1 to 4 have data that combines observations from different clones grouped together. Because there is no specific information on the 12 clones, all scientific data is lost, and no external reader can see what exactly is going on. All figures should be split into the 4 groups described, and for each group, all individual clone data should be provided. The figure legend should include how many mice were used to obtain data for each clone, and how many repeats were done. Without this information, the paper is just a 'cartoon'. With this data, this reviewer is convinced the paper could have a big impact and could open the door even for collaborative work, as it seems the authors have a very valuable unique collection in their hands. But, for that the scientific data has to be there so it is clear for every clone what is exactly happening.

Interpretation issue:

The authors started with 12 stabilates and selected from every stabilate a single clone. In the discussion, they 'walk' backwards and suggest that the individual virulence of each clone, could reflect different levels of virulence of human infections. In order to do that, a crucial experiment is missing. In order to make such conclusion one should take different clones from one and the same , and show that within a stabliate, all clones have the same level of virulence. This is actually very unlikely because it is known that every field stabilate contains a whole collection of different parasites expressing different VSGs and possibly even using different expression sites. Taking one clone per stabilate, and just assuming that this clone represent the behavior of the entire population has no scientific basis at all. Hence, any conclusion based on such assumption is juts scientifically wrong. Please remove that speculation section from the discussion because it undermines the intellectual value of the paper.

Reviewer #2: This is a nicely written manuscript describing a study of virulence and drug resistance in 12 isolates of the human-infective kinetoplastid parasite Trypanosoma brucei rhodesiense, which causes the acute East African form of sleeping sickness. In brief, the authors assessed the virulence of each isolate by infecting groups of 10 male Swiss White mice and measuring survival times, parasitemia progression and changes in body weight and packed cell volume. They also used mice to determine the sensitivity of each isolate to several commonly used HAT drugs, including Melarsoprol, Diminazene acetate, Pentamidine and Suramin, administered at doses ranging from 1 - 40 mg/kg body weight. Their main result is that despite the existence of substantial variation in virulence amongst isolates, all 12 isolates were highly sensitive to each of these drugs, even when administered at the low doses.

As I am not an epidemiologist, I am not in a position to judge the clinical significance of this work. However, I have a few concerns, possibly minor, that I would encourage the authors to address.

1.) It would be helpful to non-specialist readers if the authors included a few sentences remarking on the clinical relevance (or lack thereof) of the mouse model for virulence and drug resistance of T. brucei in humans. For example, is there any reason to expect that isolates that are exceptionally virulent in mice will be also be exceptionally virulent in humans and vice versa? The authors do demonstrate that Tbr isolates previously characterized as sensitive or resistant remain so using the mouse assays described in this paper, but similar controls are not provided for virulence.

2.) In lines 277-278, the authors remark that there is no correlation between virulence and number of passages since isolation and they cite Table 2 as evidence, but I fail to see how the information in Table 2 makes this point. Furthermore, it would appear that the very acute isolates are mostly older than the less acute isolates. Is there evidence of a reduction in the virulence of Tbr in recent years, perhaps because of enhanced genetic drift as the number of Tbr infections decreases?

3.) In lines 236-238, the authors remark that several of the mice treated with diminazene acetate died "due to causes not related to trypanosome infection." Please provide more details, e.g., what was the cause of death? In light of the small number of mice (5) used to assess resistance, it is somewhat concerning that so many of them (14) died from unrelated causes.

4) This study suggests that virulence is not a strong predictor of drug resistance for Tbr sampled over this time scale from these particular locations. However, the significance of this result is somewhat weakened by the fact that none of the tested isolates exhibited resistance. Are there data from other locations that suggest that such a correlation sometimes exists?

5.) In Table 2, Sensitive Isolate KETRI 2537 is shown as resistant to MelB at the lowest drug does (1 mg/kg) despite the fact that all 5 mice are shown as being cured. Is this a data entry error? If not, pleas explain the result.

There are several places in the text where the wording could be improved. These are listed below with the corresponding line numbers:

line 45: two subspecies

line 47: tsetse flies

line 53: two stages, namely the early (hemolymphatic) and the late (meningo-encephalitic) stage.

line 59: as well as anemia

line 72: In their study, Pyana and colleagues [13]

line 86: 6- to 8-week old

line 114: What do you mean by "ip"?

line 130: The classification of trypanosome virulence was based

line 141: determined for Melarsoprol

lines 149 and 151: 10 ml

line 160: if there are

line 170: exhibited variation

line 185: were not statistically significant

line 258: [27] does not appear to be the correct reference for this statement about Tbr genetic variation

line 259: the existence of variation in virulence among

line 261: Uganda

line 310: Our results showed a decline in body weight in the early days of infection (7 - 14 dpi) followed by a recovery except in mice infected with very acute strains.

line 312: confirms a previous observation

line 318: I'm not sure what you mean by "with days post infection"?

line 341: there is variation in virulence

line 348: Other studies are needed to confirm

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Reviewer #1: No

Reviewer #2: Yes: Jay Taylor

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29 May 2020

#

Reviewers comments Response

Why was the control group (not infected mice) not included in Table 1. Although this group is not mentioned in Materials and Methods, it is mentioned in results in lines 200 and 213. This would add more credence to the results. We concur with the reviewer’s suggestion and have therefore added the following sentence to the materials and methods section “A control group of 10 non-infected mice was included in the study and monitored for changes in PCV, body weight and survival times” ”margin lines 127-128. We have also included the control group in table 1 of the marked manuscript margin line 277-278”

In line 188, please mention the groups that the mice infected with very acute clone were being compared to. We concur with the reviewer. In the revised manuscript, this statement has been re-phrased to read as follows: “When parasiataemia data for T. b. rhodesiense clones in each virulence class were grouped together and compared, parasitaemia was significantly (p<0.001) higher in mice infected with very- acute clones, ”margin line 253-256 of the marked manuscript.

We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:

https://doi.org/10.1371/journal.pntd.0001857 https://doi.org/10.1017/S0031182017002359 https://www.afro.who.int/health-topics/trypanosomiasis-african

We have made changes to the manuscript to address the issue of overlapping texts. Where the text in question needs to be maintained, we have provided appropriate citations as follows:

1) https://doi.org/10.1371/journal.pntd.0001857.

We have modified our text to read as follows:

According to Maclean et al (2008), the pathology encountered in the acute HAT infections is characterized by elevated Tumor necrosis factor alpha (TNF-α) while that encountered in the chronic HAT infections is characterized by elevated transforming growth factor (TGF-β) (Line 66-68)

2) https://doi.org/10.1017/S0031182017002359

In the revised manuscript, the affected section now reads as follows “Each mouse’s survival time was determined on the basis of a ≥ 25% decline in PCV and consistently high parasitaemia levels of 1x109/ml for at least two consecutive days as previously described by Kamidi et al,2018 [26]” (margin lines 141-146 of the marked manuscript).The work by Kamidi et al, 2018 was done in the same laboratory in which the present study was done, hence similar methods were used to determine survival time.

3. https://www.afro.who.int/health-topics/trypanosomiasis-african

We have improved our text to read ‘’ The sensitivity of these isolates to suramin and melarsoprol is significant since currently they are the only drugs of choice recommended by WHO (2018) to treat early and late stages of Tbr HAT respectively’’. Lines 4324-436 of the marked manuscript

Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary) We acknowledge this observation by the reviewer and has hence forth corrected this in the revised manuscript. “Margin lines 8,13 and 15 of the marked manuscript”

Please upload a new copy of Figure 1 as the detail is not clear. Please follow the link for more information: http://blogs.PLOS.org/everyone/2011/05/10/how-to-check-your-manuscript-image-quality-in-editorial-manager/

A new copy of this figure has been uploaded

Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

We concur with the reviewer. Supporting information captions are now included at the end of the manuscript as suggested.

Reviewer # 1

1 Figures 1 to 4 have data that combines observations from different clones grouped together. Because there is no specific information on the 12 clones, all scientific data is lost, and no external reader can see what exactly is going on. All figures should be split into the 4 groups described, and for each group, all individual clone data should be provided. The figure legend should include how many mice were used to obtain data for each clone, and how many repeats were done. Without this information, the paper is just a 'cartoon'. With this data, this reviewer is convinced the paper could have a big impact and could open the door even for collaborative work, as it seems the authors have a very valuable unique collection in their hands. But, for that the scientific data has to be there so it is clear for every clone what is exactly happening. We appreciate this observation by the reviewer. For each of the figures 1-4, we have now provided data for each of the clones as supporting information as follows: survival (Fig 1(i)-(iv) in S1, Parasitaemia Fig 2 (i)-(iv) in S2, PCV Fig 3 (i)-(iv) in S3 and body weight Fig 4(i)-(iv) in S4 and updated the relevant sections of the text as follows: survival data (margin lines 185-203), parasitaemia (margin lines 234-256), PCV (margin lines 293-306) and body weight (margin lines 311-317) of the revised manuscript.

The number of mice used in each virulence study is now provided in the legend as was suggested by the reviewer (survival, line 203, parasitaemia, line 257, PCV, line 307 and body weight, line 318)

2 The authors started with 12 stabilates and selected from every stabilate a single clone. In the discussion, they 'walk' backwards and suggest that the individual virulence of each clone, could reflect different levels of virulence of human infections. In order to do that, a crucial experiment is missing. In order to make such conclusion one should take different clones from one and the same, and show that within a stabilate, all clones have the same level of virulence. This is actually very unlikely because it is known that every field stabilate contains a whole collection of different parasites expressing different VSGs and possibly even using different expression sites. Taking one clone per stabilate, and just assuming that this clone represent the behavior of the entire population has no scientific basis at all. Hence, any conclusion based on such assumption is juts scientifically wrong. Please remove that speculation section from the discussion because it undermines the intellectual value of the paper.

We thank the reviewer for this comments. We have addressed the reviewer’s comments in two ways:

1) Expanding on our explanation why we used cloned stabilates instead of field stabilates. In the revised manuscript, the sentence reads “We used T. b rhodesiense clones because they represent a homogeneous population of genetically identical trypanosomes thus making subsequent studies based on these clones more reproducible and reliable as previously reported (lines 359-361)

2) We have deleted reference to clinical profiles of HAT as suggested by the reviewer (margin lines 372-373).

3) We have also included the statement ’However, we recommend a similar study be carried out using the parent primary isolates (margin lines (463-465) of the revised manuscript.

Reviewer # 2

1 It would be helpful to non-specialist readers if the authors included a few sentences remarking on the clinical relevance (or lack thereof) of the mouse model for virulence and drug resistance of T. brucei in humans. For example, is there any reason to expect that isolates that are exceptionally virulent in mice will be also be exceptionally virulent in humans and vice versa? The authors do demonstrate that Tbr isolates previously characterized as sensitive or resistant remain so using the mouse assays described in this paper, but similar controls are not provided for virulence.

We concur with the reviewer’s suggestions and have therefore inserted in the statement “Studies on disease pathogenesis, parasite virulence, drug sensitivity and identification of new potential drug targets and staging biomarkers are commonly carried out in the mouse model based on its cost effectiveness, genetic similarity to humans estimated to be 97.5%, and ethical limitations of carrying out such studies in higher animal models or humans [14,15,16] .” margin lines 80 -84 of the marked manuscript

Direct comparison of parasite virulence data obtained using inbred mice (minimal host variation) and data obtained from clinical cases of HAT is difficult due to host variations in humans. Therefore, since the use of inbred mice is designed to minimize/eliminate the effect of host variations on disease outcome, it is common to infer that parasites found to be virulent in mouse model would also be virulent in humans.

No controls were provided for virulence because the idea for the current study was to compare parasite virulence with drug sensitivity patterns.

2 In lines 277-278, the authors remark that there is no correlation between virulence and number of passages since isolation and they cite Table 2 as evidence, but I fail to see how the information in Table 2 makes this point. Furthermore, it would appear that the very acute isolates are mostly older than the less acute isolates. Is there evidence of a reduction in the virulence of Tbr in recent years, perhaps because of enhanced genetic drift as the number of Tbr infections decreases? We appreciate the reviewers comment. We would, however, like to clarify that it was never our intention to state that there is no correlation between virulence and number of passages. Rather, the intended meaning was that since the study isolates had undergone only a minimal number of passages, the observed differences in virulence are likely caused by intrinsic attributes of the trypanosome. In the revised manuscript, the relevant sections reads as follows: “The majority (11/12) of the Tbr clones used in this study had undergone a minimal number of passages (1-8) passages since isolation (Table 2). The only isolate that had undergone a significant (64) number of passages (Table 2) did not exhibit notably different virulence characteristics from the other clones in the class, suggesting therefore that the observed differences in isolate virulence is an intrinsic attribute as previously reported [36] (lines 378-383) of the revised manuscript. Table 1 has been modified to show the number of passages of each trypanosome isolate (margin line 277-278). There is no data comparing virulence between older and recent Tbr isolates and there will be need to undertake such a study in future.

3 In lines 236-238, the authors remark that several of the mice treated with diminazene acetate died "due to causes not related to trypanosome infection." Please provide more details, e.g., what was the cause of death? In light of the small number of mice (5) used to assess resistance, it is somewhat concerning that so many of them (14) died from unrelated causes. We are in agreement with the reviewer and have rephrased the sentence to read as follows:…. died at 47 days post treatment and without parasitologically detectable trypanosome (Table 3), This is presumably due to pneumonia resulting from cold environment caused by a leaking water bottle. “margin lines 339-342 of the marked manuscript”

4 This study suggests that virulence is not a strong predictor of drug resistance for Tbr sampled over this time scale from these particular locations. However, the significance of this result is somewhat weakened by the fact that none of the tested isolates exhibited resistance. Are there data from other locations that suggest that such a correlation sometimes exists? We appreciate this concern raised by the reviewer. This has now been improved in the revised manuscript and reads as follows” Overall, the fact that the test isolates were all sensitive (at least 80% cure rates) to the drugs suggests there was no relationship between isolates’ virulence and their sensitivity to anti-trypanosomal drugs which is in agreement with previous studies [46,47]. In a study by Sokolova et al, [48]these authors observed that resistance to nifurtimox did not compromise parasite virulence. This is despite previous studies reporting that drug resistant trypanosome have reduced virulence [49,50]. (margin lines 444-449).

5 In Table 2, Sensitive Isolate KETRI 2537 is shown as resistant to MelB at the lowest drug does (1 mg/kg) despite the fact that all 5 mice are shown as being cured. Is this a data entry error? If not, please explain the result. We welcome this observation by the reviewer and has as a results corrected our data to read 0/5 implying all the 5 mice were resistant to MelB 1mg/kg (margin line 345-346) of the marked manuscript.

line 45: two subspecies This is now corrected as suggested by the reviewer “margin line 47 of the marked manuscript”

line 47 tsetse flies This now corrected as suggested by the reviewer “margin lines 49 of the marked manuscript”

line 53 two stages, namely the early (hemolymphatic) and the late (meningo-encephalitic) stage. This now corrected as suggested by the reviewer “margin line 55 of the marked manuscript”

line 59 as well as anemia Corrected as suggested by the reviewer “margin line 61 of the marked manuscript”

line 72 In their study, Pyana and colleagues [13] Corrected as suggested by the reviewer “margin lines 74-75 of the marked manuscript”

line 86 6- to 8-week old Corrected as suggested “margin line 93 of the marked manuscript”

line 114 What do you mean by "ip"? This is an abbreviation of intraperitoneal as described on margin line 112 of the marked manuscript

line 130 The classification of trypanosome virulence was based Corrected as suggested “margin line 138 of the marked manuscript”

line 141 determined for Melarsoprol

Corrected as suggested “margin line 151 of the marked manuscript”

lines 149 and 151 10 ml Corrected as suggested “margin lines 161, 162 and 165 of the marked manuscript”

line 160 if there are Deleted the word exist and rephrased the sentence to read ….. if there are significant differences….. “margin line 170 of the marked manuscript”

line 170 exhibited variation Corrected as suggested “margin line 182 of the marked manuscript”

line 185 were not statistically significant Corrected as suggested by the reviewer “margin line 232 of the marked manuscript”

line 258 [27] does not appear to be the correct reference for this statement about Tbr genetic variation We wish to clarify that this reference [27] is in the material and method section on determination of survival and has no reference to genetic variation (margin lines 145 -146 of the marked manuscript).Statement on genetic variation is in the discussion section and is supported by reference [30] margin line 360-361.

line 259 the existence of variation in virulence among Corrected as suggested “margin line 362 of the marked manuscript”

line 261 Uganda Corrected as suggested “margin line 364 of the marked manuscript”

line 310 Our results showed a decline in body weight in the early days of infection (7 - 14 dpi) followed by a recovery except in mice infected with very acute strains. Corrected as suggested “margin lines 422of the marked manuscript”

line 312 confirms a previous observation Corrected as suggested “margin line 426 of marked manuscript”

line 318 I'm not sure what you mean by "with days post infection"? We concur with the reviewer that this statement is ambiguous and is now deleted “margin line 432 of the marked manuscript”

line 341 there is variation in virulence Corrected as suggested “margin line 459 of the marked manuscript”

line 348 Other studies are needed to confirm

This has been rephrased to read as follows “Since our study was based on laboratory cloned T. b. rhodesiense stabilates, however, future studies should utilize the parent primary isolates”.

“ margin line 463-465 of the marked manuscript”

Submitted filename: Response to reviewers comments.docx

Click here for additional data file.

12 Jun 2020

PONE-D-20-02583R1

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

PLOS ONE

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Reviewers' comments:

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Reviewer #2: (No Response)

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Thank you for addressing the concerns flagged by this reviewer after assessment of the first version of the manuscript.

Reviewer #2: The authors have successfully addressed all of the major concerns raised in my original review and the

resulting study makes an interesting contribution to our understanding virulence and drug resistance in

African trypanosomiasis. However, there are still some minor grammatical errors and one questionable

claim in the revised manuscript, as listed below. Note that the line numbers given here refer to the

unmarked revised text in the file sent for review, not the marked revised text that appears in the second

half of the file.

line 91: The authors remark that the mouse has a "genetic similarity to humans estimated to be 97.5%",

but I don't know any way of defining genetic similarity that would give such a high number. According

to the website https://www.genome.gov/10001345/importance-of-mouse-genome, the average similarity

between the shared protein-coding genes is around 85%. Please delete or modify this claim.

line 45: A cure rate of at least 80% was achieved for all test isolates

line 48: evidence of variation in ... confirms that this variation is

line 73: in different foci differ in both their

line 87: this variation

line 143: once a week using a

line 146: The classification of trypanosome virulence

line 165: were prepared

line 179: body weight changes

line 181: The general linear model in SAS was ... between the means of the four virulence classes.

line 190: exhibited variation in

line 196: Mice infected with the acute clones had MST ranging from 18.1 +- 0.7 (KETRI 2487) to 26 +- 2.0 for

... (You don't need to keep repeating the phrase mean +- SEM for each set of numbers.)

line 207: The overall MST was 8.7 +- for the very acute clones, 21.6 +- 1.0 for the acute clones, ...

line 227: are shown in Table and Figure 2(v).

line 231: clone specific variation was observed

line 257: The pre-infection PCV data for all infected

line 258: The PCV of the infected mice declined significantly following infection when compared

line 308: at least 80% cure rates for all drug dose regimens

line 309: a few cases of relapse

line 314: without detectable parasites.

line 343: a previous report of variation in virulence

line 356: differences in isolate virulence are an intrinsic attribute

line 357: Parasitemia progression differed significantly among Tbr isolates assigned to different virulence

classes on the basis of survival time.

line 363: acute infections result from

line 371: they reveal that the majority

line 391: on the causes of body weight changes in animals infected with trypanosomes, especially after

line 413: due to the mutation or absence of a drug uptake gene

line 419: In summary, this study has found that there is variation in

**********

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Reviewer #2: Yes: Jay Taylor

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2 Jul 2020

line 91: The authors remark that the mouse has a "genetic similarity to humans estimated to be 97.5%",but I don't know any way of defining genetic similarity that would give such a high number. According to the website https://www.genome.gov/10001345/importance-of-mouse-genome, the average similarity between the shared protein-coding genes is around 85%. Please delete or modify this claim. We thank the reviewer for this observation and has modified this claim as suggested. It now reads ‘’……. genetic similarity to humans estimated to be 85 %,……..’’

Margin line 95 of the marked manuscript

line 45: A cure rate of at least 80% was achieved for all test isolates This has now been rephrased to read as suggested. Margin lines 45-46 of the marked manuscript

line 48: evidence of variation in ... confirms that this variation is We have corrected this as suggested. Margin line 50 of the marked manuscript.

line 73: in different foci differ in both their Corrected as suggested. Margin line 74 –marked manuscript

line 87: this variation Corrected as suggested. Margin line 88-marked manuscript.

line 143: once a week using a Corrected as suggested. Margin lines 144-145- marked manuscript

line 146: The classification of trypanosome virulence Corrected as suggested. Margin line 147-marked manuscript

line 165: were prepared Corrected. Margin line 166

line 179: body weight changes Corrected. Margin line 180

line 181: The general linear model in SAS was ... between the means of the four virulence classes. Corrected as suggested. Margin lines 182-183

line 190: exhibited variation in Corrected. Margin line 191

line 196: Mice infected with the acute clones had MST ranging from 18.1 +- 0.7 (KETRI 2487) to 26 +- 2.0 for... (You don't need to keep repeating the phrase mean +- SEM for each set of numbers.) In agreement with the reviewer and has deleted the repeated phrase mean +- SEM. Margin lines 197-198

line 207: The overall MST was 8.7 +- for the very acute clones, 21.6 +- 1.0 for the acute clones, Corrected as suggested. Margin lines 207-209 –marked manuscript.

line 227: are shown in Table and Figure 2(v). Corrected .margin line 229-marked manuscript

line 231: clone specific variation was observed Corrected as suggested. Margin line 233

line 257: The pre-infection PCV data for all infected Corrected. Margin 259

line 258: The PCV of the infected mice declined significantly following infection when compared Corrected as suggested. Margin line 260-261-marked manuscript.

line 308: at least 80% cure rates for all drug dose regimens Corrected. Margin line 310-marked manuscript

line 309: a few cases of relapse Corrected. Margin line 311-marked manuscript.

line 314: without detectable parasites. Corrected. Margin 316- marked manuscript

line 343: a previous report of variation in virulence Corrected. Margin line 345-marked manuscript

line 356: differences in isolate virulence are an intrinsic attribute Corrected. margin 358- marked manuscript

line 357: Parasitemia progression differed significantly among Tbr isolates assigned to different virulence classes on the basis of survival time Corrected as suggested. Margin line 359-360-marked manuscript

line 363: acute infections result from Corrected Margin line 366- marked manuscript.

line 371: they reveal that the majority Corrected. Margin line 374-marked manuscript.

line 391: on the causes of body weight changes in animals infected with trypanosomes, especially after Corrected. Margin line 393-394-marked manuscript

line 413: due to the mutation or absence of a drug uptake gene Corrected. Margin line 416

line 419: In summary, this study has found that there is variation in Corrected. Margin line 422-marked manuscript.

Submitted filename: Response to reviewer # 2 comments.docx

Click here for additional data file.

7 Jul 2020

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

PONE-D-20-02583R2

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Academic Editor

PLOS ONE

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Reviewers' comments:

16 Jul 2020

PONE-D-20-02583R2

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

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