Michele Abate

Vincenzo Salini

Eugenio Rimondi

Costantino Errani

Marco Alberghini

Mario Mercuri

Patrizia Pelotti

POST TRAUMATIC MYOSITIS OSSIFICANS : SONOGRAPHIC FINDINGS

Keywords: Myositis ossificans, Ultrasound, Soft tissue calcification, musculoskeletal system Myositis ossificans, Ultrasound, Soft tissue calcification, musculoskeletal system Myositis ossificans, Ultrasound, Soft tissue calcification, musculoskeletal system

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Sonographic findings of myositis ossificans

reports in literature [6,7,[START_REF] Kim | Myositis ossificans in psoas muscle after lumbar spine fracture[END_REF] and in most of them the diagnosis has not been confirmed by histopathology.

The aim of this paper is therefore to report the US features of PMTO in the early and mature stages, confirmed by histological examinations, in a relatively large sample of patients.

Material and methods

Fifteen patients (8 M : 7 F, mean age 31.2; SD 18,60 years, range from 12 to 65 years) referred to our Department, were retrospectively enrolled (from November 2006 to April

2009).

In these patients, PTMO was suspected on the basis of history, symptoms, physical examination and US.

US evaluation was performed using a multi -frequency (5 -12 MHz) linear array transducer (Acuson, Siemens Medical Solution, Germany).

Lesions were evaluated according to a standard protocol, using multiplanar scans; Power Doppler (PD) as well as tissue harmonic imaging was used.

The following parameters were evaluated:

1) Mass (appearance, echogenicity, margins and dimension);

2) Calcification (position, appearance, distal shadowing and dimension); 3) PD signal; 4) Relationship with contiguous structures (muscle, bone, vessels and nerves). A semiquantitative grading system was used to evaluate the neovascularization inside the area of the lesion interested by PD signal. According to this system grade (0) is no Doppler (3+++), 50 -90 % colour in the region of interest; (4++++), 90 -100 % colour in the region of interest [START_REF] Boesen | Tendinopathy and Doppler activity: the vascular response of the Achilles tendon to exercise[END_REF].

PTMO diagnosis was then confirmed by histopathology, performed at the same time on specimens obtained by means of real -time US guided needle biopsy. This procedure was carried out not only for diagnosis confirmation but also for clinical purposes (possible surgical planning). Prior to biopsy, a written informed consent was obtained from the patient.

Biopsies were done under local anesthesia and performed in sterile conditions. The needle pathway was decided on the basis of safe access route (according to the orthopaedic surgeon), avoidance of vessels and nerves (vital structures), skin to lesion distance, and also the expected definite treatment plans to avoid any possible intercompartimental contamination or seeding [START_REF] Shin | Image -guided percutaneous biopsy of musculoskeletal lesions in children[END_REF].

Under real -time US monitoring, a 13 -14 Gauge (150 mm) Tru -Cut needle was guided inside the lesions and a tissue sample was obtained.

In total, at least 4 -5 specimens were collected from the central, intermediate and peripheral layers of the lesion in order to better differentiate early from mature PTMO at histopathological examination.

Results

All the patients complained pain, local heat and swelling with decreased range of motion of the compartment affected. A palpable mass, in general harder in mature PTMO, was found in 7 cases (46,6 %).

In 10 patients (66,6 %) the lesion occurred in the thigh (8 right and 2 left), while in 5 cases (4 right and 1 left) it occurred in the arm.

A single direct blow was reported in 73,3 % patients while in the remaining 26,7 % of patients no history of trauma was reported.

The time elapsed between the injury and the US evaluation, although without significant statistical difference, was quite longer for mature PTMO (32.5 ± 17.6 days vs 17.5 ± 3.54 days in early PTMO). This wide range of values is probably due to the characteristics of the trauma and individual reactivity.

The longest and transverse mean diameters were 5.5 ± 1.7 cm (range from 9.2 to 3 cm) and 3.4 ± 0.9 cm (range from 5.3 to 1.6 cm) respectively (Tab. 1).

Mature PTMO was observed in 10 patients and early PTMO in 5. The hystopathological criteria for early PTMO were evidence of degeneration and necrosis of the damaged muscle, fibroblasts proliferation, whereas those for mature PTMO were presence of ground substance, mineralisation and bone like tissue (Fig. 1).

As common characteristics of both early and mature PTMO, US examination showed, in all cases, an oval shaped homogenous hypoechoic soft tissue mass when scanned along its long axis and rounded on transverse sonograms. Mass borders appeared regular and not infiltrative.

In all the patients, the mass was located in the muscle belly, which appeared thickened; muscle fibers were not interrupted.

Sonographic findings of myositis ossificans

The other structures adjacent to PTMO were preserved; in particular, no connections with the bone, nerves and vessels were reported.

In the early phases of PTMO, a center of less echogenicity was observed in all cases, associated with an outer sheet -like hyperechoic, not well defined, peripheral rim in three. No clear zonal demarcation or calcification was present (Fig. 2).

In the mature phases, with the increasing ossification, the peripheral rim -like calcification was more reflective with a distal acoustic shadowing in all cases. However, in three patients, the superficial calcified wall did not completely obscure the deep boundary and few internal calcific deposits were seen (Fig. 3).

Only in the early PTMO, vascularization was observed and was classified as 1+ (two cases), 2++ (two cases) (Fig. 4) and 3+++ (one case). In none of mature PTMO cases, PD signal was present (Tab. 2).

No complications (bleeding, nerves injuries, etc) apart from pain in the biopsy site were complained by the patients.

Discussion

The present study, performed on fifteen cases of PTMO, confirmed by histopathological examinations, shows that some US features (homogenous hypoechoic mass, well defined and regular borders) are common both to the early and mature stages of the disease.

The early phase is characterized by a center of less echogenicity associated with an outer hyperechoic area consistent with lamellar bone, without a clear calcification. These separate zones of varying echogenicity correspond pathologically to an outer muscular layer, an inner layer of immature osteoid and mature peripheral lamellar bone, and an innermost layer of proliferating fibroblasts, necrosis, and hemorrhage.

In the mature phase, a peripheral rim -like calcification is observed and few internal calcific deposits may be seen (presence of ground substance, mature lamellar bone, with a minor immature component of osteoid on histopathological examination).

Obviously, several intermediate stages may be visualized.

Vascularization, evaluated with a semiquantitative method [START_REF] Boesen | Tendinopathy and Doppler activity: the vascular response of the Achilles tendon to exercise[END_REF], is mainly observed in the early phase, while it progressively disappears as the lesion progresses to more mature stages.

Our results are in agreement with other authors [2, 15 -19], both for the early phase and the mature one. In particular Okayama et al. [START_REF] Okayama | Usefulness of ultrasonography for early recurrent myositis ossificans[END_REF] described, in a case of recurrent PTMO, the presence of an echogenic area, representing a soft tissue mass, with a center of less echogenicity inside and no evidence of calcification.

The diagnosis of PTMO, besides clinical history and physical examination, is based on different imaging techniques (RX, CT, bone scintigraphy, MRI and US) [2,20,21].

RX is often carried out as a first examination when a PTMO is suspected, but the findings are not specific, ranging from initially normal to the appearance of a soft tissue mass with faint

Sonographic findings of myositis ossificans

periostal new bone formation, within 1 to 2 weeks post injury [2]. Therefore, conventional radiography does not allow a clear visualization of PTMO in its early stages.

After 3 to 4 weeks, when mineralization of the osteoid occurs, and after months, when the deposition of dense bone is observed, the diagnosis may be suspected, but unconclusively [2].

It may be used to determine the size, density, and anatomical location only of calcified lesions and it may also be helpful in differentiating PTMO from osteosarcoma, since it reveals the peripheral calcification and the absence of invasion of surrounding tissue, that are characteristic of PTMO.

Bone scintigraphy with Tc 99 phosphate cannot distinguish the location of the lesion within tissue planes or provide precise information on the size and area of demarcation [22].

Furthermore, the increased uptake is non specific and musculoskeletal tumours and infection cannot be ruled out, as they may show similar uptake on the 3 -phases bone scintigraphy, as seen in early PTMO [22,23]. Therefore, this technique is not appropiate in the diagnosis of PTMO.

CT is useful only in the diagnosis of mature PTMO as it best demonstrates the typical zonal pattern of ossification [2] and clearly locates the lesion prior to surgical excision.

The MRI appearances of PTMO are variable and depend on the maturity of the lesion [24]. It is the most sensitive technique for identifying small, early lesions, but may be not specific. An important limitation is its relative inability to detect soft -tissue calcification [2].

It must be added that the above mentioned imaging techniques are expensive, invasive and often require sedation, especially in children [25]. Another disavantage is represented by the possible occurrence of allergic reaction to the contrast medium (in cases of bone scintigraphy and MRI). Therefore, these modalities are unsuitable for a serial visualization of developing lesions. In early stages, US is highly sensitive in the detection of calcific deposits in soft tissues (i.e.

prior to the development of the ossification) [START_REF] Okayama | Usefulness of ultrasonography for early recurrent myositis ossificans[END_REF] and it can be used for serial evaluation until mature calcified lesions are observed.

US also provides indicative features (in terms of echotexture, vascularization, margin, involvement of surrounding tissues, etc) to distinguish between PTMO and malignant soft tissue, even if, in some cases, further imaging modalities are needed and histopathological diagnosis from specimens obtained by excision is mandatory.

Moreover, the angulation of the US transducer can show the presence of a soft tissue plane between the calcification and the adjacent bony cortex or surrounding tissues, thus providing further features of benignity.

A careful and correct diagnosis of PTMO is important to decide for surgery. Indeed, surgery is indicated when vascularisation is completely absent and there is a total reflection of the US beam from the outer zone. Moreover, US can be used to guide the surgical excision, because it marks very well its anatomical extension.

The surgical excision is suitable when disabling pain and functional impairment are present.

On the other hand, it must be considered that only mature PTMO must be removed, because the recurrence is more likely to occur if a lesion is removed when it is metabolically active (presence of Power Doppler signal). Some strenghts and limitations of this study must be acknowledged. Compared to other papers, in which only few patients were evaluated and histological examination was not carried out, our study evaluated a quite large sample of PTMO, confirmed by histopathology.

At this regard, it must be underlined that a firm diagnosis can be obtained only by histopathological examination of the mass after excision; however, in our cases, we think that Other limitations are represented by the retrospective nature of this study and by the fact that other soft tissue masses have not been included in the comparison with PTMO; in addition, the patients were evaluated only once and were not followed up.

In conclusion, in our opinion, US can be considered as a useful tool in the PTMO evaluation because easy to perform, low -cost and safe for patients, especially for children. Methods: Fifteen patients with histological diagnosis of PTMO were enrolled.

The following US parameters were evaluated: mass; calcification; Power Doppler signal;

relationship with contiguous structures.

Results: Five patients (33.3 %) were diagnosed as early PTMO and ten (66.6 %) as mature PTMO.

As common characteristics of both early and mature PTMO, US examination showed an oval hypoechoic mass, without infiltrative borders, located in muscle belly.

In the early phases, a center of less echogenicity was observed, associated with an outer sheet -like hyperechoic peripheral rim, while, in mature phase, the peripheral calcification was more reflective and distal acoustic shadowing was observed. As common characteristics of both early and mature PTMO, US examination showed an oval hypoechoic mass, without infiltrative borders, located in muscle belly.

Conclusions

In the early phases, a center of less echogenicity was observed, associated with an outer sheet -like hyperechoic peripheral rim, while, in mature phase, the peripheral calcification was more reflective and distal acoustic shadowing was observed. Among these entities, PTMO is the most common, comprising including 60 -75 % of all cases, and may occur after a single direct blow or repeated minor traumas, although no history of trauma is observed in approximately 40 % of patients [2,4].

Conclusions

Thigh and arm are the commonest locations [5], even if some cases have been reported in the hand, neck, calf, shoulder, thorax and abdomen [6 -10]. Pain, oedema, swelling as well as palpable mass with associated decreased range of motion are usually complained by the patients [5].

Microscopically, degeneration and necrosis of the damaged muscle are seen first; afterwards the proliferation of mesenchymal cells, mineralisation and bone formation are observed.

From a clinical and radiologic point of view, the differential diagnosis includes hematoma, abscess, focal rhabdomyolysis, and malignant primary or secondary soft tissue tumors [START_REF] Damron | Diagnosis and treatment of joint -related tumors that mimic sports -related injuries[END_REF].

The imaging techniques, used to confirm PTMO, are a) conventional radiography (RX), b) bone scintigraphy, c) computed tomography (CT), d) magnetic resonance (MR) and e) ultrasonography (US) [2].

Among these imaging modalities, US is the more most suitable, because easy to perform, low cost and safe for the patient.

However, the US features of PTMO are not well established : actually, there are very few case

Sonographic findings of myositis ossificans

reports in the literature [6,7,[START_REF] Kim | Myositis ossificans in psoas muscle after lumbar spine fracture[END_REF] and in most of them the diagnosis has not been confirmed by histopathology.

The aim of this paper is therefore to report the US features of PMTO in the early and mature stages, confirmed by histological examinations, in a relatively large sample of patients.

Material and methods

Fifteen patients (8 M : 7 F, mean age 31. In all these patients, PTMO was suspected on the basis of history, symptoms, physical examination and US.

US evaluation was performed using a multi -frequency (5 -12 MHz) linear array transducer (Acuson, Siemens Medical Solution, Germany).

Lesions were evaluated according to a standard protocol, utilizing using multiplanar scans;

Power Doppler (PD) as well as tissue harmonic imaging was used.

The following parameters were evaluated:

1) Mass (appearance, echogenicity, margins and dimension);

2) Calcification (position, appearance, distal shadowing and dimension);

3) PD signal; 4) Relationship with contiguous structures (muscle, bone, vessels and nerves).

A semiquantitative grading system was used to evaluate the neovascularization inside the area of the lesion interested by PD signal. According to this system grade (0) is no Doppler This procedure was carried out not only for diagnosis confirmation but also for clinical purposes (possible surgical planning). Prior the to biopsy, a written informed consent was obtained from the patient.

Biopsies were done under local anesthesia and performed in sterile conditions. The needle pathway was decided on the basis of safe access route (according to the orthopaedic surgeon), avoidance of vessels and nerves (vital structures), skin to lesion distance, and also the expected definite treatment plans to avoid any possible intercompartimental contamination or seeding [START_REF] Shin | Image -guided percutaneous biopsy of musculoskeletal lesions in children[END_REF].

Under real -time US monitoring, a 13 -14 Gauge (150 mm) Tru -Cut needle was guided inside the lesions and a tissue sample was obtained.

In total, at least 4 -5 specimens were collected from the central, intermediate and peripheral layers of the lesion in order to better differentiate early from mature PTMO at histopathological examination.

Results

All the patients complained pain, local heat and swelling with decreased range of motion of the compartment affected. A palpable mass, in general more hard harder in mature PTMO, was found in 7 cases (46,6 %).

In 10 patients (66,6 %) the lesion occurred in the thigh (8 right and 2 left), while in 5 cases (4 right and 1 left) were observed it occurred in the arm.

A single direct blow was reported in 73,3 % patients while in the remaining 26,7 % of patients no history of trauma was reported.

The time elapsed between the injury and the US evaluation, although without significant statistical difference, was quite longer for mature PTMO (32.5 ± 17.6 days vs 17.5 ± 3.54 days in early PTMO). This wide range of values is probably due to the characteristics of the trauma and the individual reactivity.

The longest and transverse mean diameters were 5.5 ± 1.7 cm (range from 9.2 to 3 cm) and 3.4 ± 0.9 cm (range from 5.3 to 1.6 cm) respectively (Tab. 1).

Mature PTMO was observed in 10 patients and early PTMO in 5. The hystopathological criteria for early PTMO were evidence of degeneration and necrosis of the damaged muscle, fibroblasts proliferation, whereas those for mature PTMO were presence of ground substance, mineralisation and bone like tissue (Fig. 1).

As common characteristics of both early and mature PTMO, US examination showed, in all cases, an oval shaped homogenous hypoechoic soft tissue mass when scanned along its long axis and rounded on transverse sonograms. Mass borders appeared regular and not infiltrative.

In all the patients, the mass was located in the muscle belly, which appeared thickened; muscle fibers were hypoechoic but not interrupted.

Sonographic findings of myositis ossificans

The other structures adjacent to PTMO were preserved; in particular, no connections with the bone, nerves and vessels were reported.

In the early phases of PTMO, a center of less echogenicity was observed in all cases, associated with an outer sheet -like hyperechoic, not well defined, peripheral rim in three. No clear zonal demarcation or calcification was not present in all (Fig. 2).

In the mature phases, with the increasing ossification, the peripheral rim -like calcification was more reflective with a distal acoustic shadowing in all cases was observed. However, in three patients, the superficial calcified wall does did not completely obscure the deep boundary and few internal calcific deposits were seen (Fig. 3).

Only in the early PTMO, vascularization was observed and was classified as 1+ (two cases), 2++ (two cases) (Fig. 4) and 3+++ (one case). In none of mature PTMO cases, PD signal was present (Tab. 2).

No complications (bleeding, nerves injuries, etc) apart from pain in the biopsy site were complained by the patients.

Discussion

The present study, performed on fifteen cases of PTMO, confirmed by histopathological examinations, shows that some US features (homogenous hypoechoic mass, well defined and regular borders) are common both to the early and mature stages of the disease.

The early phase is characterized by a center of less echogenicity associated with an outer hyperechoic area consistent with lamellar bone, without a clear calcification. These separate zones of varying echogenicity correspond pathologically to an outer muscular layer, an inner layer of immature osteoid and mature peripheral lamellar bone, and an innermost layer of proliferating fibroblasts, necrosis, and hemorrhage.

In the mature phase, a peripheral rim -like calcification is observed and few internal calcific deposits may be seen (presence of ground substance, mature lamellar bone, with a minor immature component of osteoid on histopathological examination).

Obviously, several intermediate stages may be visualized.

Vascularization, evaluated with a semiquantitative method [START_REF] Boesen | Tendinopathy and Doppler activity: the vascular response of the Achilles tendon to exercise[END_REF], is mainly observed in the early phase, while it progressively disappears as the lesion progresses to more mature stages.

Our results are in agreement with other authors [2, 15 -19], who, in the ealry phase, described three separate zones of varying echogenicity (the "zone phenomenon) both for the early phase and the mature one. In particular Okayama et al. [START_REF] Okayama | Usefulness of ultrasonography for early recurrent myositis ossificans[END_REF] described, in a case of recurrent PTMO, the presence of an echogenic area, representing a soft tissue mass, with a center of less echogenicity inside and no evidence of calcification.

Also, the US features, relative to the mature phase, are similar to those reported in the literature.

The diagnosis of PTMO, besides clinical history and physical examination, is based on RX is often carried out as a first examination imaging modalities when a PTMO is suspected, but the findings are not specific, ranging from initially normal to the appearance of a soft tissue mass with faint periostal new bone formation, within 1 to 2 weeks post injury [2].

Therefore, conventional radiography does not allow a clear visualization of PTMO in its early stages.

After 3 to 4 weeks, when mineralization of the osteoid occurs, and after months, when the deposition of dense bone is observed, the diagnosis may be suspected, but unconclusively [2].

It may be used to determine the size, density, and anatomical location only of calcified lesions and it may also be helpful in differentiating PTMO from osteosarcoma, since it reveals the peripheral calcification and the absence of invasion of surrounding tissue, that are characteristic of PTMO.

Bone scintigraphy with Tc 99 phosphate cannot distinguish the location of the lesion within tissue planes or provide precise information on the size and area of demarcation [22].

Furthermore, the increased uptake is non specific and musculoskeletal tumours and infection cannot be ruled out, as they may show similar uptake on the 3 -phases bone scintigraphy, as seen in early PTMO [22,23]. Therefore, this technique is not appropiate indicated in the diagnosis of PTMO.

CT is useful only in the diagnosis of mature PTMO as it best demonstrates the typical zonal pattern of ossification [2] and clearly locates the lesion prior to surgical excision.

The MRI appearances of PTMO are variable and depend on the maturity of the lesion [24]. It is the most sensitive technique for identifying small, early lesions, but may be not specific. An important limitation is its relative inability to detect soft -tissue calcification [2].

It must be added that the above mentioned imaging techniques are expensive, invasive and

Sonographic findings of myositis ossificans

often require sedation, especially in children [25]. Another disavantage is represented by the possible occurrence evenience of allergic reaction response to the contrast medium (in cases of bone scintigraphy and MRI). Therefore, these modalities are unsuitable for a serial visualization of developing lesions.

In comparison Compared to the above mentioned techniques, US evaluation has several advantages.

In early stages, US is highly sensitive in the detection of calcific deposits in soft tissues (i.e.

prior to the development of the ossification) [START_REF] Okayama | Usefulness of ultrasonography for early recurrent myositis ossificans[END_REF] and it can be used for serial evaluation until mature calcified lesions are observed.

In addition US could also provides indicative features (in terms of echotexture, vascularization, margin, involvement of surrounding tissues, etc) to distinguish between PTMO and malignant soft tissue, even if, in some cases, further imaging modalities are needed and histopathological diagnosis from specimens obtained by excision is mandatory.

Moreover, the angulation of the US transducer can show the presence of a soft tissue plane between the calcification and the adjacent bony cortex or surrounding tissues, thus providing further features of benignity.

A careful and correct diagnosis of PTMO is important to decide for surgery. Indeed, surgery is indicated when vascularisation is completely absent and there is a total reflection of the US beam from the outer zone. Moreover, US can be used to guide the surgical excision, because it marks very well its anatomical extension.

Finally, when The surgical excision is indicated due to suitable when disabling pain and functional impairment are present it must be considered that. On the other hand, only mature PTMO must be removed, because the recurrence is more likely to occur if a lesion is removed when it is metabolically active (presence of Power Doppler signal).

Sonographic findings of myositis ossificans

Some strenght and limitations of this study must be acknowledged. Compared to other papers, in which only few patients were evaluated and histological examination was not carried out, our study evaluated a quite large sample of PTMO, confirmed by histopathology.

At this regard, it must be underlined that a firm diagnosis can be obtained only by histopathological examination of the mass after excision; however, in our cases, we think that reliable conclusions may be drawn, because several specimens were obtained from the different layers of the lesions under US control.

Other limitations are represented by the retrospective nature of this study and by the fact that other soft tissue masses have not been included in the comparison with PTMO; in addition, the patients were evaluated only once and were not followed up.

In conclusion, in our opinion, US can be considered as a useful tool in the PTMO evaluation because easy to perform, low -cost and safe for patients, especially in for children. (c) note that the underlying femoral shaft is not involved and 4c). The femoral cortex is interrupted. In these cases, the mass was located in the muscle belly, which appeared thickened. Muscle fibers were hypoechoic but not interrupted. M = Muscle; F = Femoral shaft

  Sonographic findings of myositis ossificansactivity; (1+), one or two tiny colour foci; (2++), up to 50 % colour in the region of interest;

  Sonographic findings of myositis ossificansCompared to the above mentioned techniques, US evaluation has several advantages.

  Sonographic findings of myositis ossificansreliable conclusions may be drawn, because several specimens were obtained from the different layers of the lesions under US control.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 1 :

 12341 Figure 1a). Packed and damaged muscle fibers (20x); 116x86mm (96 x 96 DPI)

Figure 2 :Figure 3 :Figure 4 :F o r P e e r R e v i e w Sonographic findings of myositis ossificans 16

 23416 Figure 2 : Early PTMO.

:Myositis

  US is highly sensitive in the detection of PTMO and it can be used for serial evaluation. It can provide indicative features in the differential diagnosis between PTMO and malignant soft tissue tumors. Keywords: Myositis ossificans, Ultrasound, Soft tissue calcification, musculoskeletal system Ossificans is a benign condition of heterotopic non -neoplastic bone or cartilage formation in / or adjacent to a muscle and in proximity to a bone[1, 2].Four different clinical entities have been described : a) Myositis ossificans post -traumatica (PTMO); b) fibrodysplasia ossficans progressiva (FOP); c) myositis ossificans associated with paraplegia and d) myositis ossificans circumscripta[3].

  2; SD 18,60 years, range from 12 to 65 years) referred to our Department, were retrospectively enrolled (from November 2006 to April 2009).

  Sonographic findings of myositis ossificansactivity; (1+), one or two tiny colour foci; (2++), up to 50 % colour inside in the region of interest; (3+++), 50 -90 % colour inside in the region of interest; (4++++), 90 -100 % colour inside in the region of interest[START_REF] Boesen | Tendinopathy and Doppler activity: the vascular response of the Achilles tendon to exercise[END_REF].PTMO diagnosis was then confirmed by histopathology, performed at the same time on specimens obtained by means of real -time US guided needle biopsy.

  Sonographic findings of myositis ossificansdifferent imaging techniques (RX, CT, bone scintigraphy, MRI and US)[2, 20, 21].

Figure 1 :Figure 2 :Figure 3 :Figure 4 :

 1234 Figure 1: PTMO histological examination in different stages (haematoxylin -eosin stain).

Table 1 :

 1 Demography and characteristics of PTMO lesion.Symptoms are reported in terms of pain, tenderness, swelling, decreased Range of Motion.

	Case Gender Age Location Symptoms	Injury	Time lapse (days)	Mean diameters	Histopathological results
							(Longest -Transverse; cm)
	1	M	59 Right thigh	Mild	Not traumatic	20	6 x 3	Early
	2	M	15 Right arm Moderate	Traumatic	20	5 x 2.3	Early
	3	M	13 Right thigh Moderate	Traumatic	15	9.2 x 5.3	Early
	4	M	28 Right thigh Marked	Traumatic	15	3.8 x 3	Early
	5	M	53 Right thigh	Mild	Traumatic	15	5.3 x 4.2	Early
	6	M	16 Right arm Moderate	Traumatic	20	5.6 x 4.2	Mature
	7	F	16 Left arm	Moderate	Traumatic	30	4.5 x 2.8	Mature
	8	F	30 Left thigh	Mild	Not traumatic	45	3.7 x 2.9	Mature
	9	M	17 Right thigh Marked	Traumatic	20	7.6 x 3.4	Mature
	10	F	25 Right thigh	Mild	Traumatic	30	5.3 x 4.2	Mature
	11	F	12 Right thigh Moderate	Traumatic	45	5.7 x 4.7	Mature
	12	F	36 Right thigh Marked	Not traumatic	30	5.2 x 3.6	Mature
	13	M	65 Right arm	Marked	Not traumatic	30	8.6 x 3.8	Mature
	14	F	58 Left thigh Moderate	Traumatic	30	4.5 x 2.9	Mature
	15	F	25 Right arm Moderate	Traumatic	45	3 x 1.6	Mature

Table 2 :

 2 PTMO US appearance and histopathological features

	EARLY	MATURE

structures (bone, muscle, nerve, vessel)

  The US appearance of Post -traumatic myositis ossificans (PTMO) is not well established, because only few cases have been described. The aim of this paper is to report the US features of PMTO in a quite large cohort of patients.

		Sonographic findings of myositis ossificans
	Purpose:
	F o r
		P
		e
		e r
		R
		e
		No infiltration; muscle fibers not interrupted; v i e muscle belly thickened w
		Degeneration / necrosis of the	Presence of ground
	Histopathology	damaged muscle; fibroblasts	substance; mineralization;
		proliferation	bone like tissue

  The US appearance of Post -traumatic myositis ossificans (PTMO) is not well established, because only few cases have been described. The aim of this paper is to report the US features of PMTO in a quite large cohort of patients.

	Sonographic findings of myositis ossificans
	Purpose: Methods: Fifteen patients with histological diagnosis of PTMO were enrolled.
	The following US parameters were evaluated: mass; calcification; Power Doppler signal;
	relationship with contiguous structures.
	Results: Five patients (33.3 %) were diagnosed as early PTMO and ten (66.6 %) as mature F r o PTMO.
	P
	e
	e r
	R
	e
	v i e
	w

: US is highly sensitive in the detection of PTMO and it can be used for serial evaluation. It can provide indicative features in the differential diagnosis between PTMO and malignant soft tissue tumors.

John Wiley & SonsJournal of Clinical Ultrasound


Ágnes Dencs

Andrea Hettmann

Tibor Martyin

Csilla Jekkel

Tivadar Bányai

Mária Takács

Phylogenetic Investigation of Nosocomial Transmission of 1 Hepatitis C Virus in an Oncology Ward 2 3 4 5

Keywords: hepatitis C, NS5B region, envelope region, nosocomial infection, phylogenetic analysis hepatitis C, NS5B, envelope region, nosocomial infection, phylogenetic analysis 23

published or not. The documents may come

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previous record of hepatitis C infection, tested positive for anti-HCV antibodies between 5 November 2007 and June 2008. Twelve of the newly infected patients were found to be HCV 6 RNA positive. The common origin of the infections was assumed. To investigate the 7 relatedness of the detected viral strains phylogenetic analyses were performed using 8 sequences from the NS5B and E1/E2 genome regions. A patient carrying HCV for years, was 9 also involved in the study. She was treated at the same oncology department and was 10 considered a possible infectious source. The previous HCV carrier harboured subtype 1b, 11 while all other patients were infected with subtype 1a. Sequences from the 12 newly infected 12 patients formed two groups. The viral sequences within the groups were very closely related. 13 A greater evolutionary distance was observed between the two groups; however, their 14 relatedness could be demonstrated by sequences from both regions with high statistical 15 support. The results indicated that nosocomial transmission occurred. The phylogenetic [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF] analyses suggested that the viruses originated from a common source, possibly a patient 17 carrying highly divergent variants. This presumed infectious source could not be identified in [START_REF] Dencs | Phylogenetic analysis of a 19 hepatitis C virus outbreak in a haemodialysis unit[END_REF] the course of this study. The genotype distribution of Hungarian control sequences included in 19 the analysis confirmed this conclusion, since HCV genotype 1a was found to be relatively 20 uncommon. 21 Introduction 1 2 Hepatitis C virus (HCV) is a member of the Hepacivirus genus within the Flaviviridae 3 family. HCV infection is a major public health issue worldwide. An estimated 170 million 4 people are infected chronically (WHO, 1997). Chronic infection with HCV can lead to 5 chronic liver disease, cirrhosis and hepatocellular carcinoma. In countries where the screening 6 of donors has been introduced, acute hepatitis C virus infections related to the transfusion of 7 blood or blood products and transplantation of solid organs have become rare. In developed 8 countries, injection drug use accounts for most new HCV infections [START_REF] Alter | HCV infection in the United States[END_REF], Esteban et 9 al., 2008). Other potential risk factors include birth to an infected mother, occupational 10 exposure, sex with an infected partner and commercial tattooing [START_REF] Indolfi | Perinatal transmission of hepatitis C virus infection[END_REF], 11 Henderson, 2003[START_REF] Wejstal | Sexual transmission of hepatitis C virus[END_REF][START_REF] Haley | The tattooing paradox: are studies of acute hepatitis adequate to 8 identify routes of transmission of subclinical hepatitis C infection?[END_REF]. Health-care associated HCV 12 infections also occur. Outbreaks of hepatitis C have been reported from haemodialysis units, 13 oncology and hematology wards and different inpatient and ambulatory care settings. Most of 14 these involved patient to patient transmission, occasionally affecting a large number of 15 patients (CDC, 2003[START_REF] Forns | Nosocomial transmission of HCV in the 32 liver unit of a tertiary care center[END_REF][START_REF] Pekova | Clinical course and outcome of a nosocomial 4 outbreak of hepatitis C in a urology ward[END_REF][START_REF] Sepkowitz | Risk to cancer patients from nosocomial hepatitis C virus[END_REF]. Usually, 16 infections are caused by breaches in basic infection control practices by medical staff, such as 17 the reuse of disposable needles and syringes, which can lead to the contamination of infusion 18 equipment or multiple-dose medication vials (Williams et al., 2003). Sepkowitz reviews 19 epidemics in oncology wards and he raises the possibility that HCV can be found in saliva 20 and tears, and might have been transmitted via exposure to these secretions. The possibility 21 that an infected health care worker might serve as the source of infection was examined in 22 most studies and was excluded in all cases. Blood transfusion was also excluded as the source 23 of the epidemics [START_REF] Sepkowitz | Risk to cancer patients from nosocomial hepatitis C virus[END_REF]. 24 Phylogenetic analysis of viral sequences obtained following such outbreaks can provide 1 molecular evidence for the relatedness of infections, identify or exclude the possible sources 2 and may help elucidate the events that occurred. Many molecular epidemiological studies on 3 hepatitis C outbreaks have utilized either the fast changing first hypervariable region (HVR1), 4 or partial sequences of the envelope region containing the HVR1 [START_REF] Izopet | Molecular evidence for nosocomial 22 transmission of hepatitis C virus in a French hemodialysis unit[END_REF], Silini et 5 al., 2002). The more slowly evolving NS5B region has been shown to be best suited for 6 genotyping and subtyping HCV [START_REF] Laperche | Comparison of hepatitis C virus 28 NS5b and 5' noncoding gene sequencing methods in a multicenter study[END_REF], but can also provide enough 7 information to confirm relatedness of viral strains [START_REF] Carneiro | Molecular and epidemiological study on nosocomial 8 transmission of HCV in hemodialysis patients in Brazil[END_REF]. However, better 8 resolution may be achieved by the analysis of the E2 region -including HVR1 -in some 9 cases, where sequences from the recipient are obtained shortly after infection (Bracho et al., 10 2005). Some compare or combine the approaches above, or perform quasispecies analysis of 11 envelope region sequences, a method which has significantly improved the understanding of 12 the transmission of virus variants between donor and recipient [START_REF] Esteban | Transmission of hepatitis C virus by a cardiac surgeon[END_REF], Bracho et 13 al., 2005[START_REF] Hmaïed | Determining the source of nosocomial 17 transmission in hemodialysis units in Tunisia by sequencing NS5B and E2 sequences of 18 HCV[END_REF]. Utilization of longer sequences of both regions has been 14 suggested for improving statistical support of results [START_REF] Casino | Variation of hepatitis C virus following serial transmission: multiple mechanisms of 11 diversification of the hypervariable region and evidence for convergent genome evolution[END_REF]. In this paper a case 15 is presented when phylogenetic analysis of sequences outside the first hypervariable region 16 reveal a more distant relationship between transmitted viral sequences. 17 Table 1 shows the regions and primers used for amplification and sequencing. 21 Nested primers published by [START_REF] Khan | 24 Improved detection of HCV Infection in hemodialysis patients using a new HCV RNA 25 qualitative assay: experience of a transplant center[END_REF] were used with modifications to detect the 22 conserved 5'UTR regions of all known genotypes of hepatitis C virus. Primers specific for the 23 NS5B region of the HCV genome were designed to detect genotype 1 viruses. PCR specific 24 for the envelope region was performed with primers described by [START_REF] Ray | 8 Hypervariable region 1 sequence stability during hepatitis C virus replication in 9 chimpanzees[END_REF]. The 25 PCR products were purified with Viogene PCR Advanced kit (Viogene, Taipei, Taiwan) 7 using the spin column method. Purified PCR products were sequenced using the DYEnamic 8 ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Little Chalfont 9 Buckinghamshire, UK). Purification of sequencing reactions were carried out according to the 10 ethanol precipitation protocol supplied with the kit. Reaction products were separated and 11 analyzed on a MegaBACE 1000 capillary sequencer (Amersham Biosciences, Little Chalfont 12 Buckinghamshire, UK). 13 14

Phylogenetic analysis 15

The following hepatitis C virus genotype 1a and 1b sequences were included in the 16 phylogenetic analysis: 1a isolates EU677253, AJ278830, EF032898, EU155378, FJ205869, 17 EU255968, FJ182001, EU256005, EU256035, EU362877, AF511948, AF511949, D10749, 18 EU256005, EF407444, and 1b isolates AY587844, AB435162, EF407483, AF165054, 19 AB049093, EF032894, EU155362, AJ132997, AF483269, AJ000009, AY460204, 20 AF207768, AB080299, AB016785, AJ238799. Sequences were aligned with the MultAlin 21 program [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF]. The nucleotide substitution model that described the data best was 22 chosen by ModelTest (Posada and Crandall, 1998) using PAUP* [START_REF] Swofford | PAUP*. Phylogenetic analysis using parsimony (* and other methods)[END_REF] as 23 implemented in FindModel. Phylogenetic trees were constructed by the Maximum Likelihood 24 method using PhyML 3.0 [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large 6 phylogenies by maximum likelihood[END_REF]. The starting tree was computed by 25 PhyML using BIONJ (Gascuel, 1997). Subtype 1c isolate AY651061 was used as an 1 outgroup. Bootstrap analysis with 1000 replicates was performed to confirm tree topology. 2 Evolutionary distance calculations (nucleotide substitution model: Kimura 2-parameter) were 3 carried out using MEGA software version 4.0 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics 25 Analysis (MEGA) software version 4.0[END_REF]. 4

Patient and control sequences obtained in this study were deposited in GenBank under the 5 following accession numbers: patient sequences from the NS5B region (FN666627-6 FN666641), E1/E2 region (FN667677-FN667690), control sequences from the NS5B region 7 (FN666642-FN666672), E1/E2 region (FN667691-FN667708), UTR region (FN668896, 8 FN668897). 9 The serum samples of 21 anti-HCV positive patients were tested for the presence of HCV 3 RNA using nested PCR with primers specific for the conserved 5'UTR region. Viral RNA 4 could be detected in the sera of 12 of the newly infected oncology patients. The patient with 5 previous HCV infection (patient 8) was also HCV RNA positive, thus was considered a 6 putative source patient in the possible transmission event(s). Antiviral treatment was begun in 7 the case of six HCV RNA positive patients. The other patients could not be treated because of 8 metastasis or other diseases. Details of the 13 HCV RNA positive patients are shown in Table 9 2. 10

For the purpose of genotyping and phylogenetic analysis, a segment of the NS5B region from 11 the HCV RNA positive patients was amplified and sequenced. Analysis of a 500 nucleotide 12 long segment of the NS5B region was performed using sequences obtained from the 13 13 patients of the oncology, 31 unrelated controls from different regions of Hungary and 30 14 genotype 1a and 1b isolates from GenBank (Fig. 1). A rooted tree was constructed using the 15 General Time Reversible model (gamma shape parameter: 0.397, proportion of invariant sites: 16 0.265). The tree showed that all 12 de novo infected patients were infected with subtype 1a 17 viruses. Patient 8 was infected with subtype 1b, and was excluded as a possible source patient. [START_REF] Dencs | Phylogenetic analysis of a 19 hepatitis C virus outbreak in a haemodialysis unit[END_REF] The genotype 1a sequences from the outbreak formed two groups with high statistical 19 support. Group 1 consisted of the closely related viral sequences from 10 newly infected 20 patients (mean evolutionary distance: 0.003) (Table 3a). Five sequences were completely 21 identical, and only the sequence from patient 2 differed from the others by more than 3 22 nucleotide positions. The sequences from the two other patients were clustered on a separate 23 branch (Group 2). These sequences were separated from Group 1 with a mean evolutionary 24 distance of 0.021. They differed from each other in three nucleotide positions, all three 25 changes also appearing on the amino acid level. Groups 1 and 2 were joined together on the 1 tree by a common branch, which appeared in 98% of bootstrap replicates, suggesting a 2 possible relatedness. The 12 sequences appeared as a cluster that was separated from subtype 3 1a isolates used for constructing the tree by a mean evolutionary distance (0.061) similar to 4 those observed between pairs of independent HCV isolates of the same subtype (mean for 5 subtype 1a: 0.050). 6

To see whether another, faster evolving region also supports this grouping, PCR was 7 performed to amplify a portion from the more variable E1/E2 region of the virus, which 8 included HVR1. Products could be obtained for only 12 patients. Patient 1 could not be 9 included in this part of the study, because the sample did not give a positive result with the 10 E1/E2 primer set even after several attempts. PCR products from 12 other samples were 11 sequenced. 12 The alignment of the 81 nucleotide long HVR1 sequences, often used to demonstrate 13 phylogenetic inference, from the oncology patients infected with 1a subtype strongly 14 supported the existence of Group 1 with only 5 sporadic substitutions in the 10 sequences, 4 15 of them nonsynonymous. No close relationship of the viruses infecting Group 1 patients and 16 patient 3 could be observed. Their HVR1 sequences differed in 12-15 amino acid residues, 17 similarly to unrelated control and GenBank isolates (Fig. 2). 18

However, when the complete determined sequence (positions: 887-1813 nt) was used to 19 construct a phylogenetic tree, it showed a similar picture to the one based on NS5B (Fig. 3). 20

Again, the General Time Reversible model fit the data best (gamma shape parameter: 0.597, 21 proportion of invariant sites: 0.266). The sequence from patient 8 was clustered together with 22 1b subtype HCV isolates. Among 1a isolates from the oncology the same grouping could be 23 observed with high statistical support. Table 3b shows the sequence statistics of the envelope 24 region. Sequences in Group 1 were very close genetically, the average distance within the 25 group was only 0.003. Thirteen variable nucleotide positions were observed, 5 of them within 1 HVR1. These resulted in three variable amino acid residues in the E1 protein (3/192, 1.56%), 2 4 within HVR1 (4/27, 14.81%) and one in the short flanking segment of E2 (1/80, 1.25%). 3

The mean evolutionary distance between Groups 1 and 2 (patient 3) was 0.049 on the 4 nucleotide level. In 39 nucleotide positions the occurring bases were characteristic for each 5 group, on the amino acid level 18 differences were unique. 6

The pairwise distances separating unrelated GenBank and Hungarian control sequences of the 7 same subtype were also calculated and -like in case of NS5B sequences -they were found to 8 be typically much greater than between the two patient groups (mean for subtype 1a: 0.115). 9

The NS5B and E1/E2 regions from patients 3 and 11 were sequenced twice, 3 months apart 10 (sequences marked T1 and T2). The NS5B regions were found to be unchanged, in the 11 envelope protein region, however, 2 and 3 substitutions could be observed on the nucleic acid 12 level for patients 3 and 11, respectively, causing one change in the amino acid sequence in 13 each patient. 14

The nucleotide sequences from 40 Hungarian HCV carriers determined in this study served as 15 unrelated controls, but also provided information on the genotype and subtype distribution of 16

Hungarian hepatitis C isolates. 38 of 40 viruses (95%) belonged to genotype 1 based on 17 5'UTR sequences. Subtypes within genotype 1 were determined based on NS5B and/or the 18 envelope region. Where both regions were sequenced (11 controls), the genotyping results 19 were in complete concordance. A single subtype 1a virus was found among the 38 genotype 1 20 isolates (1/40, 2.5%), all others were subtype 1b. In the case of two control patients only the 21 5'UTR region could be amplified and these viruses were genotyped based on the nucleotide 22 sequences of that PCR product. One of them was shown to belong to genotype 3, the other to 23 genotype 4. Six of the controls were local controls originating from the South-Eastern part of 24 After several patients at the oncology department of a hospital in Hungary with elevated ALT 3 levels and no previous record of HCV infection were found to be anti-HCV positive, 4 molecular epidemiological investigation of a possible nosocomial outbreak was performed. 5

The oncology ward had a high patient turnover with several thousand patients per year. They 6

were not routinely screened for hepatitis B or C markers, but blood tests including liver 7 function tests were performed regularly to control side effects of cytostatic treatment. Cases 8 of high liver function were further investigated. A notable increase in the incidence of 9 elevated ALT levels among patients returning for control was noticed in January and February 10 of 2008. Until October of 2008, a total of 45 patients were screened for anti-HCV antibodies, 11 and 21 of them were found to be positive. Twenty of them had no history of hepatitis C 12 infection. One patient had been a known virus carrier for years (patient 8), and was considered 13 a possible common source of infections. 14 Serum samples from 21 anti-HCV positive patients were sent for hepatitis C RNA detection, 15 genotyping and phylogenetic analysis. Thirteen samples were HCV RNA positive, including 16 patient 8, the long time carrier. Genotyping showed that de novo infected patients were 17 infected with subtype 1a. However, patient 8 carried a subtype 1b virus, and thus was not the 18 infectious source. Phylogenetic analysis of the NS5B region showed that the viruses of 10 19 newly infected patients were closely related (Group 1) and very likely had a common origin. 20

The viruses infecting the two other patients were similarly closely related to each other 21 (Group 2), but were clearly separated from the other 10 isolates by a distance less than usually 22 observed between unrelated strains. The twelve sequences formed a statistically supported 23 group that appeared monophyletic, which suggested they may be associated. Since NS5B 24 evolves relatively slowly, a large segment of envelope region including HVR1 was also 25 primer set. This occurred either because of a mutation in the primer binding regions, or the 2 lower sensitivity of this method. The results obtained were similar and provided further 3 evidence of a more distant, but common ancestor. GenBank controls also demonstrated that 4 even unrelated isolates may show relatively low genetic distances, especially in the NS5B 5 region. However, pairs of reference sequences with the smallest distances did not appear as a 6 monophyletic group (e.g. isolates FJ182001 and EU255975). The rapidly changing HVR1 7 supported the existence of Group 1, but the only sequences that could be obtained from Group 8 2 (from patient 3) showed no similarity to Group 1 and differed as much from Group 1 9 sequences as unrelated isolates. Two possible interpretations of these results may be 10 considered. One of these is that the common source of infections was a long time HCV 11 carrier, who carried highly divergent quasispecies. A small inoculum was probably transferred 12 from the source patient to the recipients, which may have only contained a fraction of these 13 variants. Different variants became dominant in the different recipients, and diverged further 14 from their ancestral sequence in the months between the infection and the collection of 15 samples. This may be an example of selective transmission of HCV quasispecies, which has 16 been first described in chimpanzee and in vitro experiments, where a total of three 17 chimpanzees were infected with the same HCV containing plasma. Of the quasispecies 18 present in the inoculum, the major variant became dominant in two animals and a minor one 19 in the third animal [START_REF] Hijikata | Selective transmission of hepatitis C virus in vivo and in vitro[END_REF][START_REF] Sugitani | Comparison of amino acid sequences in hypervariable region-1 20 of hepatitis C virus clones between human inocula and the infected chimpanzee sera[END_REF]. Selective transmission has 20 been also documented in humans. After a needle stick accident the minor clone in the donor 21 was found to be the most closely related to the major clone detected in the recipient (Saito et 22 al., 2004). In an outbreak at a haemodialysis unit sequences from two recipients showed 23 greatest similarity to a small subset of variants found in the source patient (Bracho et al., 24 2005). A HCV carrier cardiac surgeon infected several of his patients during surgery, and the 25 sequences recovered from the different recipients were related to different variants in the 1 surgeon [START_REF] Esteban | Transmission of hepatitis C virus by a cardiac surgeon[END_REF]. The transmission of a single or a small group of variants was 2 observed also in perinatal infection (Manzin et al., 2000). 3

The common source of the infections at the oncology ward may have been another HCV 4 carrier patient (or a health care worker) not identified by the field investigation, thus not 5 included in the analysis. This patient was probably missed by the screening for anti-HCV 6 antibodies either because of normal liver functions, or because of remission from or death 7 caused by the patient's primary disease. Several other patients infected recently at the hospital 8 with HCV could have also been missed by the investigation for the same reasons. 9

Unfortunately, there are not sufficient epidemiological data available to determine the order 10 the infections occurred and trace the patient-to-patient route of transmissions. 11 Another possible explanation of the results is that the sequences in Groups 1 and 2 are not 12 related directly. In this case the reason for the homology detected between them is that they 13 originate from a common population, and have an older common ancestor. To investigate this 14 possibility, the viruses of 40 HCV carrier control patients from Hungary unrelated to the 15 outbreak were also genotyped, including 6 local controls from within a 150 km radius of the 16 hospital. Genotyping of the controls showed that subtype 1b is predominant in Hungary in the 17 clinical material studied (92.5%), which is a similar result to that observed by Gervain 18 (Gervain et al., 2003). Only a single subtype 1a virus was found, originating from a different 19 part of the country and showing no notable sequence similarity to viruses of the oncology 20 patients. These results do not support that two cases of nosocomial transmission may have 21 occurred coincidentally from two unrelated HCV subtype 1a carriers both treated at the 22 hospital. [START_REF] Khan | 24 Improved detection of HCV Infection in hemodialysis patients using a new HCV RNA 25 qualitative assay: experience of a transplant center[END_REF] In summary the results suggest that transmission of hepatitis C virus occurred in the oncology 24 ward, where invasive procedures were frequent in the therapy of the patients. The infections 25 probably originated from a common source, although the source patient could not be 1 identified. The precise mode of transmission could not be ascertained either, but 2 contamination of a multiple-dose medication vial or infusion equipment could be suspected. 3

After the outbreak was discovered, strict infection control measures were introduced and 4 regularly monitored. After October of 2008 no more new cases were identified. These tested a large genotype 3a HCV outbreak at an oncology clinic and found that it was a single-15 source, patient-to-patient outbreak. [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF] Molecular methods have been used widely in investigation of hepatitis C virus outbreaks. In 17 the present study sequence analysis of the NS5B region gave the correct grouping of the 18 isolates under investigation with good statistical support. The observed clusters were 19 confirmed by the phylogenetic tree based on a large portion of the envelope region. It seems 20 that in many cases determining partial sequences (about 500 bases or more) of NS5B is 21 sufficient to show or exclude relatedness of virus strains as also applied in an earlier 22 investigation [START_REF] Dencs | Phylogenetic analysis of a 19 hepatitis C virus outbreak in a haemodialysis unit[END_REF]. This method has the added advantage that false negative 23 PCR results due to mutations in the primer binding regions are less likely to occur than in 24 case of the more variable E1/E2. The short HVR1 often used in phylogenetic analyses may 25

  In 2007 and 2008 several patients of the oncology department of a hospital inHungary 18 returned for treatment or control with elevated ALT levels. Patients treated at the department 19 were screened for anti-HCV antibodies, and twenty patients with no previous history of HCV 20 infection were found to be positive. The possibility of nosocomial transmission was 21 investigated by phylogenetic analyses of partial sequences from both the NS5B and E1/E2 22 regions. The study was approved by an institutional review committee and performed in 23 accordance with Hungarian law. All patients gave their informed consent in writing.[START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics 25 Analysis (MEGA) software version 4.0[END_REF] 21 anti-HCV positive patients of the oncology ward were received for the 4 detection of viral RNA, genotyping and phylogenetic analysis. Twenty patients were tested 5 positive for anti-HCV antibodies between November 2007 and June 2008 with no history of 6 previous HCV infection, and one patient had been known to be anti-HCV positive at least 7 since 1998. Serum samples were collected between March and November of 2008, when the 8 patients returned to the hospital for treatment or control. All patients were female, their 9 average age was 57.7 years (range 27-76). Samples from 40 Hungarian HCV carriers from 10 different parts of the country were used as controls (19 male and 21 female, average age 50.2, 11 range: 20-79), including 6 local controls within 150 kilometers from the hospital. All samples 12 were stored frozen at -20°C until the analysis. 13 14 Detection of HCV RNA 15 Nucleic acids were extracted from 200 µl serum using QIAamp MinElute Virus Spin Kit 16 (Qiagen, Hilden, Germany). Viral RNA was reverse transcribed using ImProm-II Reverse 17 Transcription System (Promega Corp., Madison, WI) with random hexamers and in the 18 presence of 20 units of Rnase inhibitor. 10 µl of the cDNA was used for amplifications in 19 each case. 20

  1026 nucleotide long product that contains the carboxy-terminus of the core 1 gene, the complete E1 region and a segment of the E2 region including HVR1. 2 All bands were detected in 2% agarose gel with ethidium bromide staining under UV

  within a 150 km radius of the hospital (c24, c26, c27, c38-40). They all carried

  and analyzed. Samples of only 12 of the patients gave positive PCR results with this 1

5 findings

 5 are similar to those published by Silini et al. (2002), Dumpis et. al. (2003), and 6 Maillaird et al. (2009) 7 Silini et al. (2002) observed 13 newly admitted patients with anti-HCV seroconversion in a 8 hematology ward. They excluded the role of HCV infected health care workers, and 9 transfusional origin. Patient-to-patient transmission and the role of multidose vials were 10 assumed as a cause of infection. Dumpis et al. (2003) tested 46 HCV infected patients on a 11 paediatric oncology ward. They also excluded the role of HCV carrier health care workers and 12 the role of the blood transfusion. It was concluded that infections could occur outside the 13 patients' room and may relate to some malpractice by the medical staff. Mailliard et al. (2009) 14

  when only a short time has passed since the infection. However, in this study 1 the more distant relatedness of the virus groups did not emerge from the analysis of this Gascuel O. 1997. BIONJ: an improved version of the NJ algorithm based on a simple model

Fig. 1 .

 1 Fig.1. Phylogenetic analysis of the NS5B region nucleotide sequences. The maximum likelihood tree includes 30 subtype 1a and 1b isolates from GenBank and 31 unrelated sequences from Hungarian HCV carriers. Sequences from the patients of the oncology are displayed in bold (p1-13), Hungarian control sequences are marked by 'c' and a number. Viral sequences from two patients (p3 and p11) were determined at two different time points (T1 and T2).

Fig. 2 .

 2 Fig. 2. Multiple alignment of HVR1 amino acid sequences from patients of the oncology and of selected HCV 1a isolates obtained from GenBank. Positions differing from the first sequence are shown with a black background.

Fig. 3 .

 3 Fig.3. Phylogenetic analysis of the E1/E2 region nucleotide sequences. The maximum likelihood tree includes 30 subtype 1a and 1b isolates from GenBank and 18 unrelated sequences from Hungarian HCV carriers. Sequences from the patients of the oncology are displayed in bold (p1-13), Hungarian control sequences are marked by 'c' and a number. Viral sequences from two patients (p3 and p11) were determined at two different time points (T1 and T2).

Table 1 .

 1 Regions of HCV and primers used for PCR and sequencing

	Region	Step	Annealing temperature °C	No. of cycle	Direction	Position*	Sequence
	5'UTR	Outer Nested	60 59	35 30	Sense Antisense Sense Antisense	43-68 348-322 78-103 313-288	5'-CCC TGT GAG GAA CTA CTG TCT TCA CG-3' 5'-TGC TCA TGG TGC ACG GTC TAC GAG ACC-3' 5'-TCT AGC CAT GGC GTT AGT ATG AGT GT-3' 5'-CAC TCG CAA GCA CCC TAT CAG GCA GT-3'
	NS5B	Outer Nested	58 57	40 35	Sense Antisense 9220-9203 8312-8332 Sense 8622-8636 Antisense 9187-9171	5'-GGA GKC AAT YTA CCA ATG YTG TGA C-3 5'-GCC GSD ATT GGA GTG AGT TTR AG-3' 5'-ACG GAK GCT ATG ACY AGG-3' 5'-ACY GCC CAR TTR AAG AGG TA-3'
	E1/E2	Outer Nested	58 59	35 30	Sense Antisense 2086-2064 834-859 Sense 843-868 Antisense 1868-1848	5'-GCA ACA GGG AAC CT TCCT GGT TGC TC-3' 5'-GGG CAG DBC ARR GTG TTG TTG CC-3' 5'-AAC CTT CCT GGT TGC TCT TTC TCT AT-3' 5'-GAA GCA ATA YAC YGG RCC ACA-3'

*Nucleotide positions are based on the H77 isolate (accession number: AF009606).

Table 3a .

 3a Sequence statistics based on the determined 500 base long segment from the NS5B region

			Nucleotide level			Amino acid level
	Compared sequences	Number of variable	Avg. no. of different	Mean genetic distance	Number of variable	Avg. no. of different	p-distance (SEM)
		positions	positions (SEM)	(SEM)	positions	positions (SEM)
	Within group 1	9	1.636 (0.535)	0.003 (0.001)	4	0.727 (0.354)	0.004 (0.002)
	Within group 2	3	3.000 (1.717)	0.006 (0.003)	3	3.000 (1.687)	0.018 (0.010)
	Between groups 1 and 2	19	10.455 (2.913)	0.021 (0.006)	7	1.864 (0.870)	0.011 (0.005)
	Among controls of the same subtype (1a/1b)	96 / 189	23.792 (2.518) / 31.245 (2.489)	0.050 (0.006) / 0.067 (0.006)	18 / 53	3.392 (0.872) / 5.029 (0.857)	0.021 (0.005) / 0.031 (0.005)
	Between subtype 1a controls and groups 1-2	112	28.670 (3.632)	0.061 (0.008)	23	5.643 (1.867)	0.034 (0.011)

Table 3b .

 3b Sequence statistics based on the determined 927 base long segment from the envelope region

	Legends to the figures
	Compared sequences F Number of variable positions o r Within group 1 13 P	Nucleotide level Avg. no. of different positions (SEM) 2.836 (0.805)	Mean genetic distance (SEM) 0.003 (0.001)	Number of variable positions 8	Amino acid level Avg. no. of different positions (SEM) 1.600 (0.569)	p-distance (SEM) 0.005 (0.002)
	Between group 1 and patient 3 Among controls of the same subtype (1a/1b)	53 326 / 471 e e 43.591 (6.529) 97.419 (4.815) / 118.640 (5.465) r	0.049 (0.007) 0.115 (0.006) / 0.147 (0.008)	25 92 / 162	21.000 (3.977) 34.267 (3.439) / 43.480 (3.764)	0.070 (0.014) 0.111 (0.011) / 0.161 (0.014)
	Between subtype 1a controls and groups 1-2	349	103.359 (6.426) R	0.123 (0.009)	104	41.979 (4.737)	0.137 (0.015)
			e
			v i e
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Journal of Medical Virology

Acknowledgements 5

The authors thank Zoltán Kugler for his help in data collection and epidemiological 6 investigations. The skilled technical assistance of Mrs Katalin Kulcsár, Mrs Mária Végh and 7

Mrs Erzsébet Dubszki is very much appreciated. 8

John Wiley & Sons

Journal of Medical Virology

F o r P e e r R e v i e w


Marie Rathcke Lillemark

Jan Gerstoft

Niels Obel

Gitte Kronborg

Court Pedersen

Louise Bruun Jørgensen

Tina Vasehus Madsen

Terese Lea Katzenstein

M.Sc Rathcke Marie

Lillemark

Prof. MD, DMSc Jan Gerstoft

MD, DMSc Niels

MD. DMSc Gitte Kronborg

Prof. MD, DMSc Court Pedersen

MD, DMSc Terese Lea

Katzenstein

Leo Nielsen

Hustru Karen

Margrethe Nielsens

Characterisation of HIV-1 from patients with virological failure to a Boosted protease inhibitor regimen

Keywords: Ritonavir, Resistance, PI plasma concentrations, Gag CS

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The use of highly active antiretroviral treatment (HAART) regimens with unboosted pro-2 tease inhibitors (PIs) has resulted in a high level of virological failure primarily due to 3 the development of resistant virus. Current boosted PI regimens combine successfully 4 low-dose ritonavir (r) with a second PI. The aim was to estimate the proportion of pa-5 tients, in a population based setting, who develop virological failure on a PI/r regimen. 6

Through The Danish HIV Cohort Study 1007 patients who received PI/r based treat-7 ment between 1995 and 2008 were identified. Twenty-three (2.3%) experienced vi-8 rological failure, of whom 19 (83%) started PI/r treatment before 2001. Patients from 9

Copenhagen (n=19) were selected to study the development of protease (PR) and gag 10 cleavage site (CS) mutations during PI/r treatment and PI plasma levels at the time of 11 virological failure. Three patients (16%) developed major PI resistance mutations. [START_REF] Loeb | Muta-13 tional analysis of human immunodeficiency virus type 1 protease suggests func-14 tional homology with aspartic proteinases[END_REF] Mutations in the p7/p1 and p1/p6 gag CS only developed in patients with major or 13 minor mutations in PR. Drug concentrations were low or undetectable in 10 out of the 14 19 patients. In total PR resistance mutations and low drug levels could account for 12 15 (63%) of the failure cases. In conclusion, virological failure to PI/r is a low and 16 decreasing problem primarily caused by low plasma drug levels and to a lesser extent 17 major PR mutations. Gag CS mutations did not contribute significantly to resistance 18 development and virological failure. [START_REF] Jorgensen | Prevalence of drug resistance mutations and non-B subtypes 21 in newly diagnosed HIV-1 patients in Denmark[END_REF] 20 KEY WORDS 21 Ritonavir, resistance, PI plasma concentrations, gag CS 22 INTRODUCTION 1 During maturation of the Human immunodeficiency virus type 1 (HIV-1) viral particle 2 the PR enzyme cleaves the gag polyprotein into functional proteins essential to the in-3 fectious virus [START_REF] Loeb | Muta-13 tional analysis of human immunodeficiency virus type 1 protease suggests func-14 tional homology with aspartic proteinases[END_REF][START_REF] Katz | The retroviral enzymes[END_REF][START_REF] Mammano | Resistance-associated loss of viral fitness in 6 human immunodeficiency virus type 1: Phenotypic analysis of protease and gag 7 coevolution in protease inhibitor-treated patients[END_REF]]. The 4 gag polyprotein contains five specific protease cleavage sites (CS), p24/p17, p17/p2, 5 p2/p7, p7/p1 and p1/p6 with different affinity for the enzyme. These are cleaved at spe-6 cific rates and order to obtain the mature infectious viral particle. Protease inhibitors 7 (PI) block the cleavage activity of PR by binding to the active site of the enzyme and 8 thereby hinder the maturation of the virus [START_REF] Ashorn | An inhibitor of the protease blocks the maturation of human and simian im-15 munodeficiency viruses and the spread of infection[END_REF][START_REF] Mcquade | A synthetic HIV-1 protease inhibitor with antiviral activity ar-20 rests HIV-like particle maturation[END_REF]. 9 HAART regimens with unboosted PIs have shown a high level of virological failure pri-10 marily due to the development of resistant virus. Resistance towards PIs arises when 11 specific major resistance mutations appear in the PR gene, causing a conformational 12 change in the enzyme's active site. This lowers the affinity to the PIs and allows for 13 cleavage to occur. Minor mutations develop secondarily to restore loss of viral fitness 14 and replication capacity implied by the major mutations [START_REF] Gulnik | Ki-4 netic characterization and cross-resistance patterns of HIV-1 protease mutants se-5 lected under drug pressure[END_REF]Croteau et 15 al., 1997;[START_REF] Borman | Resistance of human immunodeficiency vi-2 rus type 1 to protease inhibitors. Selection of resistance mutations in the presence 3 and absence of the drug[END_REF][START_REF] Nijhuis | Increased fitness of drug resistant HIV-1 protease as 5 a result of acquisition of compensatory mutations during suboptimal therapy[END_REF][START_REF] Mammano | Retracing the evolutionary 10 pathways of human immunodeficiency virus type 1 resistance to protease inhibitors: 11 virus fitness in the absence and in the presence of drug[END_REF]. 16

Current boosted PI regimens combine low-dose ritonavir with a second high-dose PI 17 (PI/r). This decreases the degradation rate by the cytochrome P450 enzyme in the liver 18 and increases blood and intracellular levels of the PIs [START_REF] Moyle | Principles and practice of HV-protease inhibitor pharma-23 coenhancement[END_REF] The estimated HIV-1 prevalence among adults in Denmark is 0.07% [Lohse et al., 3 2005]. About 60% of the patients are infected with subtype B virus [START_REF] Madsen | Introduction of HIV-1 into an isolated population: 23 Molecular epidemiologic study from Greenland[END_REF]4 2003]. Medical care, including antiretroviral treatment (ARV), is tax-paid and provided 5 free-of-charge to all residents of Denmark infected with HIV-1. During the study's fol-6 low-up period, national criteria for initiating HAART were HIV-1-related disease, acute 7 HIV-1 infection, pregnancy, CD4 cell count < 300 cells/µl, and, until 2001, plasma HIV-8 1-RNA >100,000 copies/ml. 9 10

Study population 11

Patients were identified through The Danish HIV Cohort Study, described earlier 12 [START_REF] Lohse | Demographics of HIV-1 infection 18 in Denmark: Results from The Danish HIV Cohort Study[END_REF]. In brief, this cohort encompasses all patients infected with HIV-1 13 seen at Danish HIV treatment clinics since 1 st of January 1995. The Danish HIV Cohort 14 Study is approved by the Danish Data Protection Agency (J-no: 2001-41-0624). 15

All patients who, as of December 31 st 2008, had received treatment with a PI/r regimen 16 including Saquinavir, Indinavir, Atazanavir or Lopinavir and with no prior failure to un-17 boosted PI were identified from the cohort. Patients with previous virological failure to a 18 non-PI containing regimen were included in the study population. [START_REF] Jorgensen | Prevalence of drug resistance mutations and non-B subtypes 21 in newly diagnosed HIV-1 patients in Denmark[END_REF] The patients with virological failure while compliant with a boosted PI regimen were 20 identified from the study population. Virological failure was defined as plasma HIV-1-21 RNA >400 copies/ml in two consecutive measurements minimum four weeks apart af-22 ter at least three months of compliance to a continuous PI/r regimen or after HIV-1-23 RNA had been <20 copies/ml. Patient files of the failure patients were scrutinised to 24 evaluate compliance. For almost all patients with virological failure or rebound the rea-25 sons for this had been discussed with the patient. Patients who had stopped treatment 26 or who reported regularly missing treatment doses were excluded. Information on the 27 For the molecular analysis of the pol and gag genes, all failure patients seen at the two 3 HIV treating centres in Copenhagen, managing 2/3 of the Danish HIV patients, were 4 selected. Plasma samples obtained at or after the time of failure, and baseline samples 5 obtained prior to any PI treatment were analysed. Failure samples taken more than two 6 months after the failure criteria had been fulfilled were only included if the patient re-7 ceived the same PI/r treatment at failure and sample date. 8

The study was approved by The Danish National Committee on Biomedical 9

Research Ethics (H-D-2009-017). Informed consent was obtained from each patient. 10 The gag sequences were obtained using BigDye Terminator v1.1 Cycle Sequencing 5 Kit (Applied Biosystems, CA, USA) with primers LF3, TVM1_R, TVM_seq1 5'-6 AAGACACCAAGGAAGC-3', TVM_seq2 5'-TTCCTAGGGGCCCTGCAATT-3', 7

TVM_seq3 5'-ACTTTTACCCATGCATT-3' and TVM_seq4 5'-8 ACATTAGAAGAAATGATG-3' (all in-house). 9

The population based sequences were run on an ABI PRISM 3100 Genetic Analyzer 10 (Applied Biosystems, CA, USA). 11 12

Sequence analysis 13

Sequences (Genbank accession numbers: AM933271-AM933291; AM933522-14 AM933542) were analysed using SeqScape v2.5 (Applied Biosystems, CA, USA) and 15 Bionumerics v5.0 (Applied Maths, Sint-Martens-Latem, Belgium) and aligned using 16

Clustal W in Geneious v4.7 (Biomatters Ltd, Auckland, New Zealand). Phylogenetic 17 neighbour-joining tree analyses for subtyping and quality control were carried out using 18 regimens with Saquinavir, Indinavir, Lopinavir or Atazanavir and had no prior failure to 3 unboosted PI. Among these 1007 patients 36 had experienced virological failure. For 4 13 of these incompliance was reported in the patient files. The remaining 23 patients 5

(2.3%) had thus experienced virological failure according to the definition described in 6 the 'Methods' section. 7

Characteristics of the study population as well as the 23 patients experiencing virologi-8 cal failure are given in Table I. Virological failure experienced by 2.7% of males (19 out 9 of 707) versus 1.3% of females ( 4 out of 300). In total 521 patients started boosted PI 10 after year 2000 of whom 4 (0.8%) experienced virological failure. This incidence was 11 higher for the years 1995 to 2000 where 3.9% (19 out of 486) starting PI/r developed 12 virological failure. For patients with a first-line regimen including PI/r 3.4% (18 out of 13 528) developed virological failure compared to 1.0% (5 out of 479) of the patients start-14 ing a non-PI/r regimen. The failure rates were similar in the groups of patients starting 15 any treatment before and after 1995 (2.3% vs. 2.2%). The incidence rates of failure for 16 each specific PI/r regimen were: boosted Saquinavir: 10.8/1000 person years of follow 17 up (PYR) (95% confidence interval 6.4-18.2) (total 1297 PYR), boosted Indinavir 18 15.8/1000PYR (95% confidence interval 7.1-35.2) (total 380 PYR), boosted Lopinavir 19 1.4/1000PYR (95% confidence interval 0.2-9.7) (total 731 PYR), and boosted Ata-20 zanavir 4.5/1000PYR (95% confidence interval 1.1-17.8) (total 449 PYR). 21

Nineteen patients attending either of the two clinics in Copenhagen experienced vi-22 rological failure on PI/r based treatment. These were included for sequence analyses of 23 the pol and gag regions at baseline and at time of virological failure (Table II). For two 24 patients (patients 16 and 17), it was not possible to obtain gag sequences from the fail-25 ure sample due to the restricted amount of stored material. 26 II). Saquinavir or Indinavir were 4 not detectable in six out of the 14 patients in whom it was measured. In two of these 5 patients ritonavir was detectable. The remaining eight patients had Saquinavir/Indinavir 6 drug concentrations in a range above the therapeutic limit. For two patients ritonavir 7 was the only drug measured, and in both cases it was undetectable, suggesting the 8 additional PI to be present at low concentrations as well. Lopinavir was detected in one 9 patient receiving this PI, but Atazanavir was below the therapeutic limit in the two pa-10 tients receiving it. For nine patients, drug concentrations were above target trough 11 value. In these patients the median viral load (VL) was 1,480 copies/ml (442-30,500) at 12 the time of virological failure. For the three patients with major resistance mutations, 13 one had plasma concentrations below target trough value and two had values above. 14 In the present study, 23 (2.3%) of Danish HIV-1 patients on PI/r treatment in the period 2 from 1995 to 2008 developed virological failure. 3

The patients included in this study received Indinavir, Saquinavir, Lopinavir or Ata-4 zanavir as part of the PI/r regimen. Virological failure during Lopinavir/ritonavir treat-5 ment without the development of PI mutations is well described [START_REF] Hicks | Long-term safety and durable antiretroviral activity 9 of Lopinavir/ritonavir in treatment-naïve patients: 4 year follow-up study[END_REF]. In 6 this study comparing previous PR regimens the failure incidence rates were higher in 7 those treated with Saquinavir and Indinavir than in those treated with Lopinavir and 8 Atazanavir (10.8/1000PYR and 15.8/1000PYR vs. 1.4/1000PYR and 4.5/1000PYR re-9 spectively). These rates were not corrected for calendar year, backbone treatment and 10 reasons of choice of treatment regimen. 11

Out of the 19 patients chosen for molecular analyses only three (16%) developed major 12 PI resistance mutations in PR explaining the viral drug escape. Three patients devel-13 oped minor mutations in the PR gene, but no major mutations were detected, that 14 could account for the virological failure observed. For the remaining patients sequenc-15 ing of PR revealed no development of resistance mutations, and the virological failure 16 must, thus, be explained by other factors. These findings are in concordance with find-17 ings from other studies, were 0-30% of virological failures to PI/r were explained by PR 18 mutations [Ananworanich et al., 2006;[START_REF] Von Wyl | Emergence of HIV-1 Drug Re-26 sistance in Previously Untreated Patients Initiating Combination Antiretroviral 27[END_REF] tions, but high VL and wild type virus due to the delayed viral response. This could ex-1 plain the observed pattern in these patients. 2 Samples were unlikely to have been collected immediately before a dose, so it is pos-3 sible that some of these patients may in fact have had too low trough levels. Drug con-4 centrations were only measured for one time point for each patient. Sequential meas-5 urements might have given a more complete picture of the actual situation. The drug 6 concentrations below target trough level are likely to be explained by poor compliance 7 even though this was considered when selecting the study population. Information on 8 compliance was not collected prospectively but evaluated looking through patient files 9 and no data for successful compliance were collected. Even for patients with no com-10 pliance issues registered these might have been contributing to the virological failure 11 observed. Drug levels equal to zero makes altered absorption or metabolism unlikely 12

explanations. 13

Baseline and failure gag sequences were analysed for seventeen patients. Gag muta-14 tion development from baseline to failure at the p7/p1 and p1/p6 CS was only seen to-15 gether with the development of major or minor PR mutations. In none of the 19 failure 16 cases analysed in this study it could be confirmed that these gag mutations had given 17 rise to resistance development and virological failure on their own. Mutations in 18 p24/p17 and p2/p7 were observed independently of PR mutations at positions 128, 19 132, 376, 381. These positions have not been linked previously to PR resistance de-20

  compliance (i.e. percentage of missed doses) had not been collected sys-1 tematically. 2

  11 PCR and sequencing 12 Sequences of the pol region were extracted from the Danish HIV Sequence Database 13 housed at the Department of Virology, State Serum Institute, which includes sequences 14 from resistance tests performed on samples from Danish HIV patients from 2000 to 15 2008. The database currently includes approximately 5000 sequences from 1200 pa-16 tients. The Danish HIV Sequence Database is approved by the Danish Data Protection 17 Agency (J-no: 2006-41-6709). 18If the sequence was not available from the database, RNA from stored plasma samples 19 was extracted using QiaAmp viral RNA mini kit (Qiagen, Hilden, Germany). The pol 20 gene including the PR and reverse transcriptase (RT) regions was amplified and se-21 quenced using a previously described in house nested RT-PCR method amplifying 22 1302 bp[START_REF] Madsen | Introduction of HIV-1 into an isolated population: 23 Molecular epidemiologic study from Greenland[END_REF].[START_REF] Nijhuis | Increased fitness of drug resistant HIV-1 protease as 5 a result of acquisition of compensatory mutations during suboptimal therapy[END_REF] Amplification of the complete gag gene was done using primers JA152[Leitner et al., 24 1995] and XM1_mod (modified from[START_REF] Gatanaga | Amino acid substitutions in 19 Gag protein at non-cleavage sites are indispensable for the development of high 20 multitude of HIV-1 resistance against protease inhibitors[END_REF] for both cDNA (65 ºC, 10 25 min; 42 ºC, 5 min; 42 ºC, 1 h, 99 ºC, 3 min) and first round of PCR (94 ºC, 10 min; (94 26 ºC, 30 sec; 65 ºC, 30 sec; 72 ºC, 1 min) 40 cycles; 72 ºC, 10 min). For the second 27 of PCR (94 ºC, 30 sec; 65 ºC, 30 sec; 72 ºC, 1 min) 30 cycles; 72 ºC, 10 min) 1 primers LF3[START_REF] Gatanaga | Amino acid substitutions in 19 Gag protein at non-cleavage sites are indispensable for the development of high 20 multitude of HIV-1 resistance against protease inhibitors[END_REF] and TVM1_R 5'-2 CCTCCAATTCCCCCTATCATTTTTG-3' (in-house) were used, generating a 1723 bp 3 PCR-fragment including all five PR cleavage sites. 4

  Geneious v4.7 (Biomatters Ltd, Auckland, New Zealand). Sequences from the Danish 19 HIV Sequence Database and subtype specific references from the Los Alamos Se-20 quence Database (http://www.hiv.lanl.gov/) were included as reference sequences. 21 Resistance associated mutations in pol according to the Stanford HIV Drug Resistance 22 Database [Liu and Shafer; 2006; last updated June 10, 2008] were identified. Full gag 23 sequences from pre-treatment and failure samples were aligned pairwise in order to 24 detect mutations in the five CS regions that had developed during treatment. CS re-25 gions were defined by five amino acids (aa) on each side of the CS. 26 27 The Danish HIV Cohort Study database had received ritonavir boosted 2

  between the baseline and failure samples was 44 months (range 24-1 124 months). Fifteen patients were infected with subtype B virus, the remaining four 2 with subtypes A, CRF-01, C and G, respectively (TableII). 3No patients had major PI resistance mutations at baseline. Patients 6, 10 and 17 de-4 veloped major PI resistance mutations during PI/r treatment, in all three cases including 5 L90M. Two had received boosted Saquinavir, and one had received boosted Lopinavir. 6Patients 7 and 13 developed only minor PI mutations. For the remaining four patients 7 with minor PI mutations at failure (2, 12, 15 and 16), these were already present at 8 baseline. From ten patients no major or minor PI resistance mutations could be de-9 tected at time of virological failure. 10Fifteen patients had mutations related to RT inhibitors (data not shown). In ten patients 11 the mutations developed during the study period and in the remaining five the muta-12 tions were already present at baseline. 13Gag sequences were analysed from 19 baseline samples and 17 failure samples. All 14 sequences deviated from the HXB2 reference sequence in one or more positions at the 15 five cleavage sites. Subtype non-B specimens exhibited more nucleotide differences 16 from the subtype B reference than the subtype B sequences. These deviations were 17 typically maintained from baseline to failure and are likely to be subtype specific poly- 18 morphisms. 19 Nine patients developed aa mutations in one or more CS between baseline and failure. 20 Most mutations and polymorphisms were observed at the p2/p7 cleavage site. The 21 most conserved site was p17/p2 with no development of mutations and only one poly-22 morph position (position 362). Only patients with major or minor mutations in PR devel-23 oped mutations at the p7/p1 and p1/p6 CS during failure.[START_REF] Katz | The retroviral enzymes[END_REF] Several insertions and deletions relative to the HXB2 reference were observed 25 throughout the gene especially in the p1 and p6 regions (data not shown). All except 26 at baseline and failure. This was a four aa insertion in p24 in the 1 failure sample of patient 12 (data not shown). 2 Drug concentrations were below target trough value in ten out of the 19 patients (Pa-3 tients 1, 4, 6, 9, 11, 12, 13, 15, 18 and 19, see Table

. 19 Previous

 19 studies have shown low PI concentrations in patients failing PI/r regimens and 20 high intra-and inter-patient variability [Zazzi, 2007; Masquelier et al., 2005; Best et al., 21 2007]. In accordance with these observations the present study found that drug con-22 centrations were below target trough value in ten out of the 19 patients. Only one pa-23 tient with measured plasma concentrations below target trough value developed major 24 mutations. In the remaining nine patients with low PI concentrations the detected VL 25was caused by replicating wild type virus. Patients who are incompliant, but take their 26 medication prior to sampling, will appear with measureable plasma drug concentra-27

velopment. 21 Variations

 21 from the reference sequence in p7/p1 and p1/p6 were observed at positions 22 428, 431, 437, 449, 451 and 452 including the 431V, 437V and 452S variants de-23 scribed in previous studies in relation to PI resistance development [Zhang et al., 1997; 24 Verheyen et al., 2006; Nijhuis et al., 2007; Myint et al., 2004; Malet et al., 2007]. These 25 variants were present at baseline as well as at the time of virological failure. They were 26 therefore most likely present during the period of viral suppression observed in all pa-27 the criteria and did thus not prevent viral suppression. Previous 1 studies have shown that certain CS mutations might contribute directly to PI resistance 2 by improving cleavage efficiency by wild type PR [Verheyen et al., 2006; Nijhuis et al., 3 2007; Prabu-Jeyabalan et al., 2004]. In the present study it could not be confirmed that 4 this was of any significance for the limited amounts of virological failure cases ob-5 served. 6 In conclusion, virological failure is infrequent in patients on a PI/r based regimen. The 7 proportion of patients failing PI/r treatment has decreased during the years 1995-2008. 8 PR resistance mutations and low drug levels could account for 12 (63%) of the failure 9 cases observed. The analyses of gag sequences did not confirm a significant role of 10 gag CS mutations in resistance development among the limited amount of failure pa-11 tients with virological failure to PI/r. The use of regimens evaluated in the present study 12 has decreased over the years, and are only used for a minority of present day patients 2007. Lack of major resistance mutations in HIV-1 protease at failure of 4 boosted protease inhibitor-based firstline antiretroviral treatment. Reviews in antivi-5 ral therapy 2: 4-12 6 7 Zhang YM, Imamichi H, Imamichi T, Lane HC, Falloon J, Vasudevachari MB, Salz-8 man NP. 1997. Drug resistance during Indinavir therapy is caused by mutations in 9 the protease gene and in its gag substrate cleavage sites. J Virol 71:6662-6670. 10

for molecular analysis to study the development of PR and gag 18 mutations during PI/r treatment as well as the plasma concentration levels of the PIs at 19 the time of virological failure. 20

	1 1	have been suggested as an explanation for the low plasma concentrations and subse-PATIENTS AND METHODS
	2 2	quent virological failure seen in some patients [Zazzi, 2007]. Setting
	3	Since gag is the natural substrate for PR, mutations in or around the gag cleavage
	4	sites have been linked to PI resistance development. Some studies assign a compen-
	5	satory fitness role to gag CS mutations [Zhang et al., 1997; Doyon et al., 1996; Bally et
	6	al., 2000], whereas others found resistance development based exclusively on muta-
	7	tions in gag [Verheyen et al., 2006; Nijhuis et al., 2007; Prabu-Jeyabalan et al., 2004].
	8	Some studies have looked in gag outside the CS and found non-CS mutations leading
	19 9 10 11 12 13 14 15 16 17	which to improved viral replication [Gatanaga et al., 2002; Myint et al., 2004; Aoki et al., F o r P e e r R e v i e 2009]. Ghosn et al., 2009, suggested that the emergence of certain gag CS mutations could predict virological failure. Though the exact role of gag CS and non-CS mutations is yet to be determined, it is widely accepted that specific mutations such as A431V and I437V in p7/p1 and R452S in p1/p6 can contribute to continued viral replication in the presence of PIs [Zhang et al., 1997; Verheyen et al., 2006; Nijhuis et al., 2007; My-int et al., 2004; Malet et al., 2007]. The purpose of the present study was to estimate the proportion of patients, in a popu-lation based setting, who develop virological failure on a PI/r based regimen. A group of o r P e e r R e patients was selected F v i e
	20	has proven to decrease the risk of virological failure and resistance development [Coo-w w
	21	per et al., 2003; Lima et al., 2008].
	22	A previous study [Ananworanich et al., 2006] found a low prevalence (3.8%) of virologi-
	23	cal failure in patients on PI/r regimens. Von Wyl et al., [2007], found that only 30% of
	24	PI/r failure patients harboured resistance mutations and Ananworanich et al., 2006,
	25	found no resistance mutations in nine patients with PI/r failure. The major reasons for
	26	failure on PI/r regimens in the absence of resistance have not been determined, though
	27	theories such as altered absorption and metabolism of the PIs in patients failing PI/r
		4 5

Journal of Medical Virology

ACKNOWLEDGEMENTS 1

The authors would like to thank "the Danish Medical Research Council" and "Fhv. Di-2

Legat for Laegevidenskabelig 3

Grundforskning" for research grants. The study was furthermore supported by an unre-4 stricted grant from ABBOTT Laboratories (Copenhagen, Denmark).