Darren George

Norman Craig

Thomas Wdj Reid

Kirsty G Martin

Janice S Davidson

Peter C Hayes

J Simpson

Darren G N Craig

Thomas Wdj

Dr Kenneth J Simpson
email: k.simpson@ed.ac.uk

The systemic inflammatory response syndrome (SIRS) and sequential organ failure assessment (SOFA) scores are effective triage markers following paracetamol (acetaminophen) overdose

Keywords: Acetaminophen, liver failure, acute, liver transplantation, Systemic Inflammatory Response Syndrome, multiple organ failure ALF, acute liver failure, OLT, orthotopic liver transplantation, SIRS, systemic inflammatory response syndrome, HE, hepatic encephalopathy, SOFA, Sequential Organ Failure Assessment, RRT, renal replacement therapy, NAC, N-acetyl cysteine, IQR, interquartile range, ROC, receiver operator characteristic, DOR, diagnostic odds ratio, CI, confidence intervals

  The systemic inflammatory response syndrome (SIRS) and sequential organ failure assessment (SOFA) scores are effective triage markers following paracetamol overdose

METHODS

Analysis of 100 single time-point paracetamol overdoses admitted to a tertiary liver centre, with subsequent prospective validation of identified thresholds. Individual laboratory samples were correlated with the corresponding clinical parameters in relation to time post-overdose, and the daily SOFA and SIRS scores calculated.

RESULTS

A total of 74 (74%) patients developed the SIRS, which occurred significantly earlier in patients who died (n=21) compared with spontaneous survivors (n=53, p=0.05). The SIRS occurred in 70 (70%) patients by 96 hours post-overdose, with a 30% mortality rate; compared with 0% mortality in the 30 non-SIRS patients (p=0.001). Median SOFA scores were significantly higher in non-survivors at 48 (p=0.009), 72 (p<0.001), and 96 hours (p<0.001). A SOFA score >7 during the first 96 hours post-overdose predicted death/transplantation with a sensitivity of 95.0 (95% CI 78.5-99.1) and specificity of 70.5 (95% CI 66. 3-71.6). A validation cohort of 38 single time-point paracetamol overdoses confirmed the extremely high negative predictive value of both the SIRS and SOFA thresholds.

CONCLUSIONS

The absence of either a SOFA score >7 or a SIRS response during the first 96 hours following paracetamol overdose could improve triage and reduce transfers of lower risk patients to tertiary liver centres. Paracetamol-induced ALF is strongly associated with the development of the systemic inflammatory response syndrome (SIRS) and a subsequent cascade of complications including hepatic encephalopathy (HE), coagulopathy, and multiple organ dysfunction syndrome. [START_REF] Rolando | The systemic inflammatory response syndrome in acute liver failure[END_REF] In human studies, development of the SIRS following paracetamol-induced hepatotoxicity has been associated with both an increased risk of worsening HE grade, [START_REF] Vaquero | Infection and the progression of hepatic encephalopathy in acute liver failure[END_REF] and with poorer overall outcome, irrespective of the presence of infection.(3) Existing prognostic systems, such as the King's College Criteria (KCC), utilise features of HE and extrahepatic organ injury to predict a hopeless prognosis without OLT, but their use has been criticised for their relatively low negative predictive value [START_REF] Renner | How to decide when to list a patient with acute liver failure for liver transplantation? Clichy or King's College criteria, or something else?[END_REF] and by the inherent requirement for multiorgan failure to have developed before considering definitive treatment.

Several studies have therefore examined the prognostic accuracy of multiorgan failure assessment scores such as the Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE) II scores to predict outcome following paracetamol hepatotoxicity, [START_REF] Mitchell | Earlier identification of patients at risk from acetaminophen-induced acute liver failure[END_REF][START_REF] Cholongitas | King's criteria, APACHE II, and SOFA scores in acute liver failure[END_REF][START_REF] Schmidt | Prognostic implications of hyperlactatemia, multiple organ failure, and systemic inflammatory response syndrome in patients with acetaminophen-induced acute liver failure[END_REF] but these studies have tended to apply the prognostic test in question at the point of hospital admission, rather than in relation to the time from overdose. [START_REF] Craig | Systematic review: prognostic tests of paracetamolinduced acute liver failure[END_REF] This inevitably introduces temporal confounding, since many patients present to hospital following paracetamol overdose due to psychological, rather than physical, morbidity. Therefore, a better understanding of the temporal evolution of the SIRS and multiorgan failure following paracetamol overdose could improve prognostication in this condition, and could aid early identification of high-risk patients. To our knowledge, no studies exist which examine the temporal relationship between the initial hepatotoxic insult (i.e. paracetamol overdose) and development of the SIRS and multiorgan failure. The aims of this study were to examine these temporal changes in a cohort of patients who required tertiary level care after taking a single intentional paracetamol overdose. It was hypothesised that the absence of a SIRS response or extrahepatic organ injury following overdose would be associated with a low risk of in hospital mortality, and that the SIRS and SOFA scores could therefore act as quantitative triage instruments to identify those patients at lower risk of requiring emergency OLT. We specifically excluded patients with staggered overdoses, or overdoses taken accidentally in an attempt to relieve pain, due to the confounding temporal and diagnostic problems associated with these overdoses. [START_REF] Gyamlani | Acetaminophen toxicity: suicidal vs. accidental[END_REF] Paracetamol overdose was defined as at least 2/3 of: a history of ingestion of potentially toxic amounts of paracetamol (>4g/day); detection of paracetamol in the serum >10mg/L; or a serum alanine aminotransferase (ALT) level> 1000 IU/L within 7 days of a history of paracetamol ingestion irrespective of the serum paracetamol concentration.(1) All 138 patients fulfilled all of these three criteria. Severe acute liver injury was defined as sudden deterioration in liver function with associated coagulopathy in the absence of a history of chronic liver disease, whilst the term ALF (i.e. fulminant liver failure) was restricted to those patients developing HE. [START_REF] O'grady | Acute liver failure: redefining the syndromes[END_REF] Guidelines for accepting patients from referring admitted to the SLTU are managed using a standard protocol as previously described, [START_REF] Simpson | The utilization of liver transplantation in the management of acute liver failure: comparison between acetaminophen and non-acetaminophen etiologies[END_REF] the main goals of which have remained similar over the duration of this cohort study. The KCC are used in this unit and throughout the UK to determine patients who will most likely die without OLT. [START_REF] O'grady | Early indicators of prognosis in fulminant hepatic failure[END_REF] Since the purpose of this study was not to develop transplantation listing criteria, death and OLT were considered equivalent when undertaking survival analysis.

LABORATORY PARAMETERS

Retrospective analysis of all hospital electronic laboratory records was performed to obtain all standard haematological, biochemical, and coagulation samples obtained for each patient during the first 10 days of admission. Each sampling time point was noted from either the clinical notes and/or the laboratory receipt, and the corresponding haemodynamic and clinical parameters for each sampling time point retrieved from the clinical notes. These data were then individually compared against the reported time from overdose for each patient, and the corresponding laboratory and clinical values recorded in a dedicated database.

SIRS

The presence of the SIRS was defined as two or more of: temperature <36°C or >38°C, heart rate >90 beats/minute, leucocyte count <4 x 10 9 /L or >12x 10 9 /L, and tachypnoea > 20 breaths/minute or PaCO 2 <4.3 kPa. [START_REF] College | /Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.[see comment][END_REF] The daily presence or absence of the SIRS, and the number of the SIRS components fulfilled, were retrospectively calculated for each patient by correlating the daily leucocyte count with the most abnormal temperature

SOFA SCORE AND ORGAN FAILURE

Organ failure was assessed using the SOFA score, which assesses six organ systems: hepatic; renal; coagulation; cardiovascular; respiratory; and central nervous; and provides a graded score from 0-4 points for each organ system, table 1.(16) The SOFA score was calculated for each 24-hour period following overdose, using the most deranged values for each parameter in each 24 hour period. Organ failure was defined as a SOFA score ≥3 points for the organ concerned (table 1). Due to potential confounding, SOFA scores were not calculated following the administration of platelet transfusions. Optimal thresholds for predicting death or liver transplantation were derived from receiver operator characteristic analysis.

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The accuracy of the SOFA score is compared with the modified King's College Hospital poor prognostic criteria (KCC). The modified SOFA score excludes the hepatic failure component.

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Association between SIRS and SOFA scores

We then explored whether those patients with high SOFA scores also manifested a SIRS response. By 96 hours post-overdose, the median (+/-IQR) SOFA score in those patients who manifested a SIRS response was significantly higher (8 (5-15)) compared with those patients with no SIRS at that point (5 (3-6.25), p=0.0011). There was a strong correlation between these two variables (Spearman's r=0.474, p<0.0001), as shown in figure 2, with those patients who died tending to have both a SIRS response and a higher SOFA score than spontaneous survivors.

Prospective validation of multiorgan failure scores

The prognostic accuracy of the SIRS and SOFA thresholds outlined above was then examined in a prospectively collected cohort of paracetamol-induced acute liver injury

DISCUSSION

This study has explored the temporal relationship between paracetamol overdose and the development of systemic inflammation and organ failure. Development of the SIRS and extrahepatic organ failure are both strongly associated with adverse clinical outcomes, and both SOFA and SIRS scores are highly sensitive markers of poor outcome following paracetamol overdose. A SOFA score ≤7 during the first 96 hours post-overdose is highly predictive of spontaneous survival, with a risk of death of <2%, and these patients also have a low risk of requiring specialist intracranial pressure monitoring or renal support. Due to the high negative predictive values of both SOFA and SIRS, these scores may have a future role as 'gatekeepers' when considering patient transfer to tertiary liver centres.

Predicting individual prognosis accurately following paracetamol overdose is fraught with difficulty.( 17) For a predictive model to be clinically useful, it should be easy to use, accurate, reproducible, and accepted by clinical staff. Both SOFA and SIRS scores are dynamic tests, which can be rapidly recalculated throughout admission, and are attractive as prognostic indicators due to their widespread use in intensive care units and their ease of use. Recently, the SOFA score was demonstrated to outperform liver-specific prognostic scores in predicting outcome amongst a cohort of critically ill chronic liver disease patients. [START_REF] Das | Cirrhotic patients in the medical intensive care unit: early prognosis and long-term survival[END_REF] Whilst both the SOFA and SIRS scores have similarly high negative predictive values following paracetamol overdose, the SOFA score is more attractive as a quantitative triage tool as, unlike the SIRS, it is an ordinal rather than dichotomous variable, and therefore a rising SOFA score may help to identify deteriorating patients. Although neither SIRS nor SOFA were originally developed to predict outcome, the high sensitivity of these scores makes them attractive as a means of identifying all those high-risk paracetamol overdose patients who might die without transplant, although their relatively lower specificity suggests that they should not replace the KCC as definitive listing criteria. Additionally, there are some specific limitations of both SOFA and SIRS scores that should be addressed.

The SIRS does not incorporate a hepatic failure component at all, whilst the hepatic failure component (serum bilirubin) of the SOFA score lacks specificity or the ability to differentiate acute from chronic hepatic dysfunction. Indeed, of all the 6 organ failure components combined in the SOFA score, only the hepatic failure element was not associated with an increased risk of death, a finding in keeping with several previous studies of this scoring system. [START_REF] Moreno | The use of maximum SOFA score to quantify organ dysfunction/failure in intensive care. Results of a prospective, multicentre study. Working Group on Sepsis related Problems of the ESICM[END_REF][START_REF] Janssens | Evaluation of the SOFA score: a single-center experience of a medical intensive care unit in 303 consecutive patients with predominantly cardiovascular disorders. Sequential Organ Failure Assessment[END_REF] Future studies should validate whether the modified SOFA score outlined above, which excludes hepatic organ failure, is a more accurate triage marker in this setting than the original SOFA score.

The principal strength of this study lies in its novel evaluation of SIRS and SOFA scores in relation to the timing of overdose, rather than in relation to the time of admission or the onset of HE. The initial symptoms of ALF are often vague, and as a result there may be a significant delay between the overdose and subsequent hospitalisation. Additionally, some patients present as a result of the psychological consequences of their overdose, rather than as a result of physical morbidity. Thirdly, since the majority of previous studies of prognostic variables of paracetamol-induced ALF have been performed at academic referral centres following patient transfer from smaller hospitals,(9) attempting to apply a prognostic score at the time of 'hospital admission' is considerably hindered by patient heterogeneity. Equally, accurately applying a prognostic score at the onset of HE may be problematic since the presence or absence of HE is subjective, and subtle encephalopathy may be wrongly interpreted as resulting from alcohol or narcotic withdrawal. We have therefore attempted to minimise clinical heterogeneity by selecting single time point overdoses, and have examined the evolution of systemic inflammation and organ failure in relation to this fixed time point.

Using this approach, we demonstrate that the prognostic accuracy of both SIRS and SOFA scores are enhanced compared with previous reports which applied these markers at either the time of hospital admission or the onset of HE. [START_REF] Craig | Systematic review: prognostic tests of paracetamolinduced acute liver failure[END_REF] We recognise that this approach is However, we recognise that this study has several important limitations. Clearly, the results of this study are not directly applicable to patients who have taken a staggered overdose, an increasingly recognised subtype of paracetamol overdose, [START_REF] Craig | Overdose pattern and outcome in paracetamol-induced acute severe hepatotoxicity[END_REF] or in cases where accurate timings of the overdose are unavailable. Although we have examined the applicability of multiorgan failure scores in relation to time from overdose, patients access treatment at different time points in their clinical evolution, which may influence the development of the SIRS or organ failure. A further caveat is that some patients were transferred to the SLTU from a number of surrounding hospitals, each of which may have had different management protocols, clinical expertise, and referral thresholds following paracetamol overdose. Whilst the SLTU is the single referral point for all severe paracetamol overdoses in Scotland, we recognise the possibility of selection bias and that some patients with severe organ failure secondary to paracetamol overdose may not have been transferred to the SLTU. [START_REF] Blair | Deaths from paracetamol poisoning in Scotland, impact of the Scottish Liver Transplantation Unit[END_REF] We also recognise that not all of the patients in this study developed HE, and therefore ALF. However, predicting the development of HE can be equally problematic, and the mere absence of HE following severe acute liver injury does not preclude the development of other complications such as acute kidney injury. [START_REF] Pakravan | Renal injury at first presentation as a predictor for poor outcome in severe paracetamol poisoning referred to a liver transplant unit[END_REF] A potential application for SIRS and SOFA scores may be as gatekeepers to tertiary level care. In the SLTU, transfer criteria are based broadly upon guidelines previously published by the British Society of Gastroenterology. [START_REF] Devlin | Indications for referral and assessment in adult liver transplantation: a clinical guideline[END_REF] These guidelines recognise that "A F o r P e e r R e v i e w significant proportion of cases will make an unremarkable recovery without intensive measures but this level of unnecessary transfer is justified if the potential for transplantation, in what is often a narrow window of time, is to be realised". Due to the rapid progression of paracetamol-induced ALF and the risks involved in transferring patients with HE or with incipient cerebral oedema, up to 50% of UK liver centre patients never develop HE.

Developing sensitive markers to identify high-risk patients at an earlier time point, prior to HE developing, could accelerate safe patient transfer, but equally the high negative predictive value of the multiorgan scores outlined above could effectively rule out the need for emergency OLT by identifying those patients highly likely to survive with supportive care alone, thereby potentially avoiding patient transfer.

In conclusion, this retrospective cohort study of 100 severe acute paracetamolinduced acute liver injury patients has evaluated the incidence and temporal kinetics of both the SIRS and SOFA scores following single time point paracetamol overdose, and demonstrates that these markers are highly sensitive at identifying patients with a poor prognosis. The high negative predictive value of these markers was validated in a prospective cohort. Both the SIRS and SOFA scores deserve further evaluation and external validation as potential gatekeepers to improve clinical decision making models following paracetamol overdose.

  ) overdose remains the main aetiological factor responsible for acute liver failure (ALF) in the developed world.(1) The only effective treatment for the condition remains emergency orthotopic liver transplantation (OLT).[START_REF] Craig | Review article: the current management of acute liver failure[END_REF]

  Since its inception in 1992, the Scottish Liver Transplantation Unit (SLTU) has prospectively collected and maintained a dedicated database of all acute liver injury patients admitted to the unit. Data recorded in this database includes details of patient history, clinical examination, and laboratory results along with therapeutic interventions, including intensive care admission, need for renal replacement therapy (RRT), or inotropic support. Where possible the paracetamol preparation, number of tablets, type (whether intentional, accidental, single time point or staggered) and timing of overdose, delay to presentation, and use of N-acetyl cysteine (NAC) are all recorded. The cohort analysed were 100 consecutive single time point intentional (defined as a paracetamol overdose (>4g) taken at a single defined time point with the objective of self-harm) paracetamol overdoses admitted to the SLTU with acute liver injury between April 2003 and February 2009, and a prospectively collected cohort of 38 patients admitted between March 2009 and July 2010. Ethical approval for the study was obtained from the Scotland 'A' Research and Ethics Committee.

  on previously published criteria from the British Society of Gastroenterology and remained unchanged over the time course of the study.(12) Patients

  recorded during each corresponding day of admission. Respiratory rate was only included for non-ventilated patients, as the majority of patients in our unit are electively mechanically ventilated following the development of grade III or IV HE.

  µmol/l (mg/dL) ≤110 (≤1.2) 110-170 (1.2-1.9) 171-299 (2.0-3.4) 300-440 (3.5-5.0); urine output ≤500 ml/day >440 (>5.0); urine output <200 ml/day Hepatic: bilirubin, µmol/l (mg/dL) for at least one hour (doses given are in µg/kg per minute).

  temporal accuracy of the patients' overdose history, but previous studies have suggested that the patient history is usually reliable following paracetamol overdose.[START_REF] Waring | Does the patient history predict hepatotoxicity after acute paracetamol overdose?[END_REF] Heterogeneity is further reduced by the single centre nature of this study, where both criteria for patient admission and clinical management protocols have remained unchanged during the time course of the study.

Figure 1 .

 1 Figure 1. Association between mortality rate and the number of organ failures identified by the Sequential Organ Failure Assessment (SOFA) score at (A) 48, (B) 72, and (C) 96 hours following single time point paracetamol overdose.

Figure 2 .

 2 Figure 2. Association between Sequential Organ Failure Assessment (SOFA) scores and the number of Systemic Inflammatory Response Syndrome (SIRS) components fulfilled by 96 hours following single time point paracetamol overdose.

Table 1 The Sequential Organ Failure Assessment (SOFA) score

 1 FiO 2 , fractional inspired oxygen; MAP, mean arterial pressure; PaO 2 , arterial oxygen tension; A total of 100 patients (46 male, 54 female) admitted to the SLTU between April 2003 and February 2009 were included in the study. During this period, a total of 347 patients had been admitted, of whom 111 were classified as 'non-paracetamol' cases, 62 patients had taken a staggered paracetamol overdose, 45 patients had overdosed on paracetamol

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Statistical Analysis

Statistical analysis was performed using SPSS (SPSS 16.0, Chicago IL, USA) and Graphpad Prism (GraphPad Software Inc., La Jolla, CA). Data values are presented as median +/-interquartile range (IQR) or percentages unless otherwise stated. When undertaking survival analysis, death and OLT were considered equivalent. Receiver operating characteristic (ROC) analysis was used to identify optimum threshold values to discriminate non-survivors. Continuous data were compared using either analysis of variance or the Kruskal-Wallis test if inter-group variances were unequal, with post-hoc Dunn's testing used to compare selected groups. Categorical data were analysed using Chisquared tests or Fishers exact test. Bonferroni corrections were undertaken for repeated measures. Missing data were treated in a last observation carried forward manner. Results were considered statistically significant when p <0.05. accidently, and in the remaining 29 cases the details of the paracetamol overdose were unclear. The median patient age of the 100 included patients was 34 (IQR 24-43) years. A total of 83 patients were transferred to the SLTU from outlying health boards, at a median time of 50 (36-66.5) hours following overdose. The remaining patients were admitted to the SLTU from wards within the Royal Infirmary of Edinburgh or from local hospitals. The median ingested paracetamol dose was 25 (17.5-41) g. All 100 patients received NAC treatment at their local hospital, at a median time from overdose of 22.75 (9.5-42.25) hours.

A total of 47 (47%) of patients had taken a mixed overdose. As shown in table 2, none of these demographic parameters or potential confounders differed significantly between survivors and patients who died/required OLT. Other baseline demographic and clinical characteristics of the paracetamol study group are outlined in table 2.

Table 2 . Demographics of study population and details of paracetamol overdose.

 2

	Date are presented as median (+/-interquartile range) or percentages as appropriate

transplanted patients who met the KCC, a total of 13 patients were excluded from listing for OLT due to active and resistant alcohol dependence (11 patients) and active intravenous drug abuse (2 patients). A total of 4 patients were listed but subsequently deemed medically unfit to survive OLT, whilst a further two patients showed clear signs of spontaneous recovery and were delisted prior to an organ becoming available. On admission patients who later died or were transplanted were significantly more coagulopathic (PT 80 (53-103) s vs. 48 (33-70) s, p=0.002), had lower serum albumins (30

[START_REF] Blair | Deaths from paracetamol poisoning in Scotland, impact of the Scottish Liver Transplantation Unit[END_REF][START_REF] Pakravan | Renal injury at first presentation as a predictor for poor outcome in severe paracetamol poisoning referred to a liver transplant unit[END_REF](25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)

g/L vs. 34 (31-37) g/L, p=0.013), and had significantly higher serum creatinine levels (174 (118-239) µmol/L vs. 94 (75-145) µmol/L, p=0.006) compared with spontaneous survivors. A significantly greater proportion of patients who died or were transplanted required multiple organ support, such as RRT, inotropic support, and mechanical ventilation (Supplementary Table

1

). SIRS and temporal relationship to overdose

  Examination of the cumulative incidence of the SIRS revealed that by 48 hours post SIRS response (Fisher's p=0.020). By 72 hours post-overdose, a total of 59/86 (68.6%) patients had developed a SIRS during admission, with a mortality rate of 28.8%, compared with a mortality rate of only 3.7% (1/27 patients, Fisher's p=0.007) in patients without a SIRS at this point. By 96 hours, the cumulative incidence of the SIRS had risen to 70 (70%) patients, with a mortality rate of 30% in this group; compared with a mortality rate of 0% in the 30 patients without the SIRS (Fisher's p=0.001). The sensitivity, specificity, negative predictive values, and DORs of the SIRS occurring by each time point post-overdose are shown in

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overdose, a total of 27/40 (67.5%) patients had mounted a SIRS response, with a mortality rate in the SIRS group of 44.4%, compared with 7.7% for those patients not mounting a

table 3 .

 3 In total, 74 (74%) of patients developed the SIRS post-overdose, but this occurred significantly earlier following overdose in patients who died (median 43.0 (28.5-

	61.0) hours, n=21) compared with patients who survived (60.0 (36.5-81.0) hours, n=53,

p=0.05). In contrast, the presence of the SIRS at the time of hospital admission, or at the development of HE, resulted in reduced sensitivity and DORs compared with the values obtained at 48, 72, and 96 hours post-overdose (table

3

). The number of SIRS components fulfilled following overdose was significantly related to increasing mortality at 48 (p=0.047), 72 (p=0.0057), and 96 (p<0.0001) hours post-overdose.

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Table 3 . Predictive accuracy of the presence of the Systemic Inflammatory Response Syndrome (SIRS) following single time point paracetamol overdose.

 3 The SIRS was defined as the presence of ≥2 SIRS components in any 24-hour period. The accuracy of the SIRS is compared with the modified King's College Hospital poor prognostic criteria (KCC).

	CI, confidence intervals; HE, hepatic encephalopathy; SLTU, Scottish Liver Transplantation Unit

A previous report also evaluated SOFA scores following paracetamol overdose, and reported that a SOFA score >8 early after admission, or >12 at the onset of HE, were the

Table 4 . Predictive accuracy of the Sequential Organ Failure Assessment (SOFA) score following single time point paracetamol overdose.

 4

	Time from overdose (hours)	Threshold SOFA score	N/ deaths	SOFA >threshold / deaths	Sensitivity (95% CI)	Specificity (95% CI)	Negative predictive value (95% CI)	Diagnostic Odds Ratio (95% CI)
	48	>4	37/12	25/11	91.7 (61.5-99.8)	44.0 (24.4-65.1)	91.7 (70.2-98.5)	8.6 (1.0-77.5)
	72	>6	84/18	40/17	94.4 (72.7-99.9)	65.2 (52.4-76.5)	97.7 (90.4-99.6)	31.8 (4.0-254.2)
	96	>7	92/15	35/14	93.3 (68.1-99.8)	72.7 (61.4-82.3)	98.2 (92.8-99.7)	37.3 (4.6-301.8)
	Up to 96	>7	98/20	42/19	95.0 (78.5-99.1)	70.5 (66.3-71.6)	98.2 (92.3-99.7)	45.4 (7.2-278.7)
	48 (Modified SOFA)	>3	37/12	19/11	91.7 (61.5-99.8)	56.0 (34.9-75.6)	94.4 (79.9-99.0)	23.4 (3.1-160.4)
	72 (Modified SOFA)	>4	84/18	33/17	94.4 (72.7-99.9)	75.8 (63.6-85.5)	98.0 (91.9-99.6)	53.1 (8.2-332.1)
	96 (Modified SOFA)	>4	92/15	38/14	93.3 (68.1-99.8)	70.1 (58.6-80.0)	98.1 (92.3-99.7)	31.7 (4.9-196.7)
	Up to 96 (Modified SOFA)	>5	98/20	37/19	95.0 (78.7-99.1)	76.9 (72.8-78.0)	98.4 (93.0-99.7)	63.3 (9.9-391.4)
				KCC +ve/
	Modified KCC	N/A	100/21	deaths	81.0 (65.0-90.7)	93.7 (89.4-96.3)	94.9 (90.6-97.5)	62.9 (15.7-251.3)
				22/17

  CI, confidence intervals; HE, hepatic encephalopathy; SLTU, Scottish Liver Transplantation Unit; N/A, not-applicable

	SOFA score and outcome
	Univariate analysis of the individual components of the SOFA score at 72 and 96
	hours post-overdose was performed to identify those components which best predicted an
	adverse outcome (Supplementary table 2). All the organ failure components except the
	hepatic failure element were strongly associated with mortality, so a modified SOFA score,
	which excluded points for hepatic failure, was computed. ROC analysis of this modified
	F o r SOFA score showed an area under the curve of 0.81 (95% CI 0.67-0.95), 0.90 (95% CI 0.84-0.97), and 0.91 (95% CI 0.84-0.98) at 48, 72, and 96 hours post-overdose respectively. F o r
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	R e specificity of 76.9 (95% CI 72.8-78.0), and negative predictive value of 98.4 (95% CI 93.0-R 99.7) (table 4). e
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A modified SOFA threshold score of >3 at 48 hours, and >4 at 72 hours, improved upon the specificity of the original SOFA score at both these time points without affecting sensitivity. A modified SOFA threshold score of >5 at any point during the first 96 hours following overdose was highly predictive of death or OLT, with a sensitivity of 95.0 (95% CI 78.7-99.1),

.

  All 7 patients who died or underwent emergency OLT manifested a SIRS response, had a SOFA score >7, and had a modified SOFA >5 within 96 hours of overdose. The specificity of the two SOFA predictors was 77.4 (95% CI 70.5-77.4) and the specificity of the SIRS was 74.2 (95% CI 67.2-74.2).

			Variable (n= 38)		Value
			Sex (male/female)	16/22 (42.1%/57.9%)
			Age (years)		35 (26.5-44.5)
			Received NAC in referring hospital		38 (100%)
	Admission laboratory	parameters	F o r WCC (x10 9 /L) Platelets (x10 9 /L) Creatinine, µmol/L (mg/dL) ALT (IU/L) Bilirubin, µmol/L (mg/dL) PT (seconds)	8.4 (6.5-11.4) 122 (71-192) 117 (61-241) (1.3 (0.7-2.7) 5198 (2974-7978) 81 (32-113) (4.7 (1.9-6.6) 41 (29-73)
		Ever encephalopathic (ALF) P Grade 3-4 HE encephalopathy e Mechanical ventilation e KCC met r SIRS response by 96 hours post-overdose	18 (47.4%) 12 (31.6%) 13 (34.2%) 9 (23.7%) 15 (39.5%)
		R SOFA score >7 by 96 hours post-overdose	14 (36.8%)
	e Modified SOFA score >5 by 96 hours post-overdose	14 (36.8%)
			Overall outcome Transplanted Spontaneous survival Died without transplantation	v i e w	4 (10.5%) 31 (81.6%) 3 (7.9%)

patients admitted to the Royal Infirmary of Edinburgh between March 2009 and July 2010. A total of 38 patients were included, and their demographic details, admission laboratory parameters, and clinical outcomes are outlined in table 5

Table 5 . Validation cohort of 38 single time point paracetamol overdoses

 5

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Raph L Hamers
email: r.hamers@pharmaccess.org.

Rebecca Oyomopito

Cissy Kityo

P Phanuphak

Margaret Siwale

S Sungkanuparph

Francesca Conradie

R Oyomopito #

ME Botes N Kumarasamy

T Sirisanthana

S Abdallah

Pck Li

N Ngorima

P Kantipong

A Osibogun

Ckc Lee

W S Stevens

A Kamarulzaman

I Derdelinckx

Yma Chen

R Schuurman

M Van Vugt

T F Rinke

Cohort Profile: the PharmAccess African (PASER-M) and the TREAT Asia (TASER-M) Monitoring Studies to Evaluate Resistance -HIV Drug Resistance in sub-Saharan Africa and the Asia-Pacific

Keywords: HIV drug resistance, antiretroviral therapy, sub-Saharan africa, Asia, genotype, monitoring HIV drug resistance, antiretroviral therapy, sub-Saharan Africa, Asia, mutations, genotype, monitoring

whether they are published or not. The documents may come

According to World Health Organization (WHO) estimates, 33.4 million people were infected with the human immunodeficiency virus (HIV) type 1 globally at the end of 2008 [START_REF]AIDS epidemic update[END_REF]. Sub-Saharan Africa and Asia are the two regions having the highest HIV prevalence with 22.4 million and 4.7 million people infected, respectively [START_REF]AIDS epidemic update[END_REF]. Over the 5 years prior, access to combination antiretroviral therapy (ART) in low and middle-income countries increased 10fold to 4 million people, providing coverage to 42% of those in need [START_REF]the health sector : progress report[END_REF]. Several studies have reported significant reductions in HIV-related morbidity and mortality for individuals with access to treatment in these regions [START_REF] Braitstein | Mortality of HIV-1-infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries[END_REF][START_REF] Ivers | Efficacy of antiretroviral therapy programs in resource-poor settings: a meta-analysis of the published literature[END_REF][START_REF] Jahn | Population-level effect of HIV on adult mortality and early evidence of reversal after introduction of antiretroviral therapy in Malawi[END_REF]. In resource-limited settings, to facilitate the rapid expansion of access to ART, WHO recommends a standardized, public health approach [START_REF]Antiretroviral therapy for HIV infection in adults and adolescents: Recommendations for a public health approach[END_REF]. This is in contrast to the individualized patient management strategies in developed countries, based on routinely available diagnostic monitoring [START_REF] Keiser | Public-health and individual approaches to antiretroviral therapy: township South Africa and Switzerland compared[END_REF]. Standardized first-line ART regimens consist of a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dual nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) backbone, available in some countries as generic fixed-dose combinations [START_REF]Antiretroviral therapy for HIV infection in adults and adolescents: Recommendations for a public health approach[END_REF]. Recommended second-line regimens combine a ritonavir-boosted protease inhibitor (PI) with two previously unused and/or recycled NRTIs [START_REF]Antiretroviral therapy for HIV infection in adults and adolescents: Recommendations for a public health approach[END_REF].

Routine HIV viral load monitoring is not generally available in resource-limited countries and treatment failure is frequently identified based on immunological definitions and/or the occurrence of clinical events [START_REF]Antiretroviral therapy for HIV infection in adults and adolescents: Recommendations for a public health approach[END_REF]. Virological breakthrough may be detected late while the failing regimen is continued, thus facilitating the acquisition and accumulation of drug resistance-associated mutations [START_REF] Gupta | Virological monitoring and resistance to firstline highly active antiretroviral therapy in adults infected with HIV-1 treated under WHO guidelines: a systematic review and meta-analysis[END_REF]. Drug-resistant HIV variants may compromise the effectiveness of subsequent lines of treatment and their transmission to newly infected individuals has severe public health consequences [START_REF] Rahim | Geographic and temporal trends of transmitted HIV-1 drug resistance among antiretroviral-naive subjects screening for two clinical trials in North America and Western Europe[END_REF][START_REF] Wensing | Prevalence of drug-resistant HIV-1 variants in untreated individuals in Europe: implications for clinical management[END_REF]. To date, ART programs have been implemented without accompanying HIV drug resistance (HIVDR) monitoring.

Monitoring studies are hampered by the lack of a molecular laboratory infrastructure required for genotypic resistance testing, logistical challenges related to maintaining specimen integrity in remote settings and the high costs of testing [START_REF] Hamers | The status of HIV-1 resistance to antiretroviral drugs in sub-Saharan Africa[END_REF]. Challenges to scaling up ART in resource-limited countries, such as absence of routine virological monitoring and limited choices of drug regimen, advocate for the development of a global public health framework to monitor and prevent the emergence of HIVDR and thus maximize long-term ART effectiveness [START_REF] Bennett | The World Health Organization's global strategy for prevention and assessment of HIV drug resistance[END_REF].

HIV-1 subtype B is the predominant viral subtype in North America, Western Europe and Australia, and antiretroviral (ARV) drugs have been developed on this subtype. However, in sub-Saharan Africa and Asia, the genetic diversity in HIV subtypes and circulating recombinant forms (CRFs), resulting from recombination between subtypes within a dually infected person, is extensive [START_REF] Hemelaar | Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004[END_REF]. Although current evidence is limited, some reports have suggested that the propensity to develop HIVDR and the spectrum of mutations that emerge during drug selective pressure, may differ across subtypes and CRFs [START_REF] Dumans | Synonymous genetic polymorphisms within Brazilian human immunodeficiency virus Type 1 subtypes may influence mutational routes to drug resistance[END_REF][START_REF] Gonzalez | Impact of nelfinavir resistance mutations on in vitro phenotype, fitness, and replication capacity of human immunodeficiency virus type 1 with subtype B and C proteases[END_REF][START_REF] Kantor | Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration[END_REF][START_REF] Martinez-Cajas | Role of genetic diversity amongst HIV-1 non-B subtypes in drug resistance: a systematic review of virologic and biochemical evidence[END_REF]. Viral heterogeneity may, therefore, have implications for rates of disease progression and patient response to ART, warranting further study of inter-subtype differences in mutational pathways to resistance.

To help assess the extent of HIVDR in sub-Saharan Africa and Asia, LAASER (Linking African and Asian Societies for an Enhanced Response to HIV/AIDS; http://www.laaser-hivaids.org) has established a collaborative bi-regional program with the primary aim of increasing regional capacities for the monitoring of HIVDR. PharmAccess Thirteen clinical sites and 2 reference laboratories in 6 African countries (Kenya, Nigeria, South Africa, Uganda, Zambia and Zimbabwe) are collaborating on PASER-M (Figure 1a). [START_REF] Mcgowan | Cohort Profile: Caribbean, Central and South America Network for HIV research (CCASAnet) collaboration within the International Epidemiologic Databases to Evaluate AIDS (IeDEA) programme[END_REF]. Sites are generally government or university-based clinics and hospitals or private clinics, situated in major cities and other urban areas. Those with ethics approvals prior to June 2010 are shown in Figure 1b. ART has been available in Asia for more than 10 years, even in less resourced countries in the region and all TASER-M clinical sites have onsite virologic testing.

Details of the PASER-M collaborating clinical sites are summarized in

Clinical sites follow their national guidelines to assess eligibility for ART initiation in accordance with WHO recommendations [START_REF]Antiretroviral therapy for HIV infection in adults and adolescents: Recommendations for a public health approach[END_REF]. Genotypic resistance testing on PASER and TASER clinical specimens are performed in TAQAS-certified genotyping laboratories [START_REF] Land | TREAT Asia Quality Assessment Scheme (TAQAS) to standardize the outcome of HIV genotypic resistance testing in a group of Asian laboratories[END_REF].

Laboratories are encouraged to become accredited members of the WHO/HIVResNet HIV Drug Resistance Laboratory network [START_REF] Bertagnolio | World Health Organization/HIVResNet Drug Resistance Laboratory Strategy[END_REF]. Population-based nucleotide sequencing of the HIV protease (PR) and partial reverse transcriptase (RT) gene regions is performed on plasma specimens which have HIV RNA >1 000 copies/ml. Plasma is obtained from blood collected in EDTA-tubes which is locally stored at -80°C and, if required, batch-shipped on dry ice to a genotyping laboratory. PASER-M genotypic testing is concentrated in two central reference laboratories, and thus depends on a robust cold-chain and web-based specimen tracking system for managing specimen shipments. Approximately half of TASER-M clinical sites have an on-site or local genotyping laboratory. Most genotyping laboratories amplify viral sequences using inhouse methods, based on assembled commercially available assay components and laboratory-specific sequencing and amplification primers. One TASER laboratory uses the commercial kit TruGene (Bayer HealthCare, Tarrytown, NY, USA). The online Stanford interpretation system is used by most laboratories to identify drug resistance-associated mutations [START_REF] Rhee | Human immunodeficiency virus reverse transcriptase and protease sequence database[END_REF]. Resistance genotyping is generally performed retrospectively (i.e. not real- (web appendix). PASER and TASER sequences are submitted to the ViroScore database (Advanced Biological Laboratories SA, France) for data storage.

What do PASER-M and TASER-M cover and who is included in the sample?

The monitoring studies are multi-center prospective cohort designs with sequential patient enrolment. Patient eligibility criteria are listed in Table 3. (PASER-M) or CDC class (TASER-M), viral hepatitis co-infection status, CD4 count, HIV viral load, HIV subtype, drug adherence, ARV history and ART regimen as covariates. Predictors of drug resistance will be assessed using logistic regression models. Incidence of drug resistance will be summarized using person-years methods and Kaplan-Meier plots. Cox proportional hazards models will be used to assess risk factors associated with developing drug resistance.

What is the anticipated attrition?

The actual attrition in PASER-M and TASER-M cannot currently be accurately estimated because the duration of follow-up in the databases is still limited. In sub-Saharan Africa patient retention in routine ART programs has been estimated at 61.6% [START_REF] Rosen | Patient retention in antiretroviral therapy programs in sub-Saharan Africa: a systematic review[END_REF] to 66.8% (2) at 24 months on ART, attrition being mainly due to loss of follow-up and early death [START_REF] Rosen | Patient retention in antiretroviral therapy programs in sub-Saharan Africa: a systematic review[END_REF].

Therefore, in PASER-M the original site-specific sample size was calculated accounting for 20% loss to follow up and 25% mortality after 24 months. Attrition is expected to vary between sites, as a result of differences in patient populations, care provided and provisions for tracing lost to follow-up. TASER-M sites are generally sourced from the ongoing TREAT Asia HIV Observational Database (TAHOD) [START_REF] Zhou | The TREAT Asia HIV Observational Database: baseline and retrospective data[END_REF]. Loss to follow-up for TAHOD was 6.9/100 person-years for the 12-month period from September 2007 -September 2008. Since TASER-M monitors specified outcomes, we speculate that TASER-M follow-up will be similar to TAHOD or better. 4. For first-line patients, the median age was 36.8 years (IQR: 31.3-42.6) and 58% were women. HIV exposure was predominantly reported as heterosexual contact. More than 60% had advanced disease (classified as WHO stage III or IV) and 37% had pre-therapy CD4 counts of less than 100 cells/µL. Across all 13 sites, median baseline CD4 counts of first-line patients were <200 cells/µl (site median 135 cells/µL, range: 93 -191). Median baseline HIV viral load was 4.9 log 10 copies/ml (IQR: 4.2-5.5). The most frequently prescribed first-line regimens were based on NNRTIs (99.7%), i.e. efavirenz (EFV) and nevirapine (NVP) at 60% and 40%, respectively. First-line dual NRTI backbones were predominantly lamivudine (3TC)/zidovudine (AZT) (37%), emtricitabine (FTC)/tenofovir (TDF) (34%) and 3TC/stavudine (d4T) (26%). Overall, 67% of patients started a 3TC-containing first-line regimen. Among patients initiating first-line ART, 95% (n=2 598) reported to be ARV-naïve and 5% (n=138) had previous ARV experience, which included ART (n=60), mono/dual therapy (n=6), singledose NVP for prevention of mother-to-child transmission of HIV (PMTCT) (n=35), combination therapy for PMTCT (n=19), and unspecified (n=22). Compared with ARV-naive first-line patients, ARV-experienced first-line patients had higher median CD4 counts (177 vs. 133 cells/µL, p<0.0001), were younger (median 34.7 vs. 37.0 years, p<0.0001) and were more likely to be female (76.1% vs. 57.5%; p<0.001). Other baseline characteristics did not differ between ARV-naïve and ARV-experienced patients.

What has been found

For the 249 (8.3%) patients switching to second-line ART, the median age was 38.6 years (IQR: 32.9-44.2) and sex was equally distributed. HIV exposure was predominantly heterosexual contact and 48% of patients had advanced disease (classified as WHO stage III or IV). Median CD4 count was 125 cells/µL (IQR: 46-196). Median pre-switch HIV viral load was 4.1 log 10 copies/ml (IQR: 3.2-5.0). Ritonavir-boosted lopinavir (LPV) was the PI used almost exclusively (98%). HIVDR in sub-Saharan Africa [START_REF] Hamers | The status of HIV-1 resistance to antiretroviral drugs in sub-Saharan Africa[END_REF]. Baseline HIVDR data from Lusaka, Zambia has recently been published [START_REF] Hamers | HIV-1 drug resistance mutations are present in 6 percent of persons initiating antiretroviral therapy in Lusaka, Zambia[END_REF]. International presentations have summarized preliminary baseline HIVDR mutations and subtype distributions [START_REF] Hamers | Cohort Profile: The PharmAccess African Studies to Evaluate Resistance Monitoring Study (PASER-M) -HIV-1 Drug Resistance in sub-Saharan Africa[END_REF][START_REF] Hamers | Prevalence of drug-resistant HIV-1 variants at initiation of standard first-line HAART in Africa[END_REF]. 4. For ARV-naïve first-line patients, the median age was 36.5 years. Almost two thirds of patients were male and HIV exposure was predominantly heterosexual contact. More than one third of patients were classified as CDC class C and almost half of patients had pre-therapy CD4 counts less than 100 cells/µL. Median baseline HIV viral load was 5.0 log 10 copies/ml (IQR: 5.4-6.8) and the most common first-line regimens were based on NNRTIs (85.6%). Excluding 14 (2.4%) patients on a randomized clinical trial with a blinded NNRTI component, NVP was more commonly prescribed than EFV at 56% and 42%, respectively. First-line dual NRTI backbones were predominantly 3TC/d4T (47%), 3TC/AZT (29%) and 3TC/TDF (10%). For first-line PI regimens, the favored NRTI backbone was TDF/ FTC (33%) compared to 3TC/d4T (30%) or 3TC/AZT (18%). For the ritonavir-boosted PI component, atazanavir (ATZ) was only slightly favored over LPV at 43% vs 41%, respectively. Overall, 542 (91.4%) of ARV naive patients started a 3TC-containing first-line regimen. The 10 PMTCT patients received perinatal prophylaxis of AZT/3TC/NVP (n=7), AZT (n=2) or AZT/NVP (n=1) for between 14 and 102 days and all were prescribed AZT/3TC/NVP as first-line regimens.

As shown in

TASER-M

For the 25 (3.4%) second-line patients, 22 (88%) were of thai ethnicity and the median age was 36.5 years (IQR: 32.4 -41.9). Females were in the majority (64%), HIV exposure was

What are the main strengths and weaknesses?

Programs that monitor national and regional levels of primary and secondary HIVDR contribute to evidence-based recommendations to inform treatment guidelines and provide feedback on the success of HIV treatment and prevention programs. PASER and TASER, with TAQAS, are developing capacity in sub-Saharan Africa and the Asia-Pacific for coordinated HIVDR monitoring and genotypic laboratory testing. The study protocols are harmonized with the WHO HIV Drug Resistance Prevention Survey protocol [START_REF] Jordan | World Health Organization surveys to monitor HIV drug resistance prevention and associated factors in sentinel antiretroviral treatment sites[END_REF]. An important strength is the large number of patients and sites participating, representing a diverse spectrum of patient populations, clinic types, ART regimens and HIV subtypes.

Opportunities exist to investigate the impact of drug resistance on HIV natural history, rates of disease progression and response to treatment in non-B subtypes. Data from genotypic resistance testing will also provide insight into the population genetics and dynamics of transmitted HIVDR in the region. site are not necessarily representative of the site, country or region. Second, data quality depends on the completeness of clinical information captured through routine patient care. In PASER-M, data may have been collected under varying conditions, since some sites had no or limited research experience at study initiation. Third, at some sites, study initiation was delayed by several months due to the time required for contract negotiation, IRB study approval and, in TASER-M, procurement of FWAs. After study initiation, recruiting the required number of patients within the recommended 18 month period proved difficult for some sites, due to asymptomatic patients not seeking care or treatment, cost of medication or low-prevalence in their setting. Fourth, HIVDR monitoring activities in resource-limited countries in sub-Saharan Africa are limited by high costs of laboratory testing. To address this challenge, PASER has initiated a public-private consortium, called Affordable Resistance Test for Africa (ART-A), which aims to evaluate affordable test algorithms for the detection and interpretation of HIVDR for use in laboratories and clinics (http://www.arta-africa.org).

How can I collaborate? Where can I find out more?

Ownership of individual site data remains with the contributing site. Sites are represented by their principal investigators on the respective PASER and TASER Steering Committees.

Research is to be the subject of peer-reviewed publications and analysis priorities are driven by a concept sheet process. Both studies accept concept proposals from external researchers for review, if submitted in collaboration with one or more of the site principal investigators. The PASER and TASER protocols contribute data under the LAASER partnership (http://www.laaser-hivaids.org) and TASER also contributes data to IeDEA [START_REF] Mcgowan | Cohort Profile: Caribbean, Central and South America Network for HIV research (CCASAnet) collaboration within the International Epidemiologic Databases to Evaluate AIDS (IeDEA) programme[END_REF].

Collaborating sites are also encouraged to make an appropriate subset of their data available to Ministry of Health in their respective country in order to contribute to local efforts in monitoring HIVDR. Questions regarding participation, research concepts or requests for data should be sent to Tobias Rinke de Wit, email: t.rinkedewit@pharmaccess.org (PASER), or Thida Singtoroj, email: thida.singtoroj@treatasia.org (TASER).

  Scheme (TAQAS) which is an ongoing assessment program to build genotyping laboratory

  all participants. Details of the genotyping laboratories are summarized in Table2

  The main study objectives are to assess prevalence and incidence of HIVDR, mutational patterns and factors associated with HIVDR in persons initiating first-line ART or switching to a second-line regimen due to treatment failure under routine circumstances. Participants are required to sign informed consent prior to study enrolment and must initiate or switch ART within 30 days (PASER-M) or 181 days (TASER-M) following baseline specimen collection. Regimen switch due to treatment failure may be determined clinically, as assessed by disease progression, immunologically, by CD4 cell count, or virologically, by HIV viral load. A single drug substitution, due to toxicity or intolerance, is not considered a regimen switch. Each site aims to recruit a total of 240 participants. Second-line participants are recruited among first-line participants failing therapy and the clinical site patient population. The recommended maximum site-specific enrolment period is 18 months. How often are participants followed-up? What data are being collected? Participants are followed up as per local standard of care guidelines. The frequency of followup visits for patients varies by site (range: every 1 to 6 months). The studies make use of clinical data collected during routine visits and recorded in medical records. HIV viral load measurement and, if HIV RNA value >1 000 copies/ml, genotypic resistance testing is performed on plasma specimens taken at baseline, prior to regimen switch due to treatment failure and at annual follow-up. For patients failing a first-line regimen, the treatment failure data collection becomes the new baseline for the second-line regimen. Annual follow-up is calculated from this point. For patients failing a second-line ART, the treatment failure data collection is the final assessment prior to the patient going off study. PASER-M clinical data are recorded on standardized hard-copy data forms which are completed at three-monthly intervals and entered in a web-based clinical data system, called the HAART Monitoring System. PharmAccess performs quality assurance measures which include (i) source data verification during 3 to 6-monthly site audits, (ii) checks to identify data entry inconsistencies or suspect data values and (iii) specimen tracking. TASER-M site personnel extract clinical data from site databases and medical records collected as part of usual care. From March 2008 to March 2009, TASER-M data were submitted electronically to NCHECR on a quarterly basis, as part of study start up, then at 6 monthly intervals. At each transfer, NCHECR performs quality assurance measures which include (i) checks to identify data entry inconsistencies or suspect data values, (ii) specimen tracking and (iii) ARV history completeness. Annually, a random 10% of TASER-M patients are selected for internal site audit where submitted data is compared to patient medical records. The studies capture standardized virologic and genotypic data at protocol determined intervals. Genotyping data consists of HIV subtype and HIVDR mutations, including insertions and deletions. TASER-M also records discordant subtypes, i.e. when the PR and RT region subtypes differ. Laboratory specimen tracking information is recorded during specimen processing, allowing assessment of pre-analytical and assay validity. Genotyping laboratories complete an annual laboratory survey which includes the dynamic range of the virologic assay used, the regions of PR and RT genome routinely sequenced and the interpretation algorithm used. Observational patient data includes demographic parameters, physical measures, Centers for Disease Control and Prevention (CDC) class (TASER-M) or WHO clinical stage (PASER-M), serology of hepatitis and syphilis (TASER-M), opportunistic infections, current ART regimen, ARV history, concomitant medications, routine laboratory parameters (including CD4 counts) and assessment of drug adherence. Main analyses will include age, sex, ethnicity, HIV exposure category, WHO clinical stage

  Patient recruitment commenced in March 2007 and was completed in September 2009.Twelve of 13 sites reached the site-specific target of 240 participants, enrolling a total of 3 005 participants. Excluding patients with protocol violations (n=16) and key data missing (n=4), 2 985 patients were included in the analysis. Of these, 2 736 (91.6%) were eligible for a first-line ART regimen and 249 (8.3%) were eligible for second-line ART due to treatment characteristics are summarized in Table

  Patient recruitment commenced in March 2007 and for the March 2009 transfer, 7 sites from Thailand, Hong Kong and Malaysia provided data. Of 773 patients, 755 (97.7%) commenced ART within 181 days of baseline specimen collection and 728 (96.4%) participants had genotypic data available. Of these, 693 (95.2%) ARV-naïve patients and 10 (1.4%) ARVexperienced patients following prior PMTCT were eligible for first-line regimens. A further 25 (3.5%) patients were eligible for second-line ART following first-line treatment failure. Patient characteristics are summarized in Table

8 ( 1 .

 81 heterosexual (92%) and 60% of patients had experienced at least one CDC class C event. Of 21 patients with CD4 counts available within 6 months of starting a secondline therapy, the median CD4 count was 197 (IQR: 109 -299). Median pre-switch HIV viral load was 4.0 log 10 copies per/ml (IQR: 3.6 -4.5). All patients were on PI-based regimens, following failure on first-line NNRTI-based regimens (median duration 30.3 months). The most commonly prescribed PI was ritonavir-boosted LPV (88%). As shown in Table5, from analysis of the 728 available viral sequences, the most common subtypes were CRF01_AE (584, 80.2%) and subtype B (111, 15.2%). Non-CRF01_AE recombinants were identified in 1%) patient specimens. For 21 (2.9%) specimens, the subtype differed between PR and RT regions suggesting dual infection or recombination. International presentations have summarized 2009 baseline HIVDR mutations and subtype distributions[START_REF] Sungkanuparph | HIV-1 drug resistance among antiretroviral-naive HIV-1-infected patients in Asia: results from the TREAT Asia Studies to Evaluate Resistance-Monitoring Study (TASER-M)[END_REF][START_REF] Oyomopito | Cohort profile: the TREAT Asia studies to evaluate resistance monitoring study (TASER-M) -HIV drug resistance in the Asia-Pacific[END_REF].Complete baseline and prospective outcome data for PASER-M and TASER-M are anticipated to become available in 2010-2013.

WFigure 1a .

 1a Figures & Tables

Table 3 .---

 3 Geographical location of PASER-M (a) and TASER-M (b) collaborating sites 233x247mm (96 x 96 DPI) Geographical location of PASER-M (a) and TASER-M (b) collaborating sites 619x464mm (72 x 72 DPI) Patient eligibility criteria for PASER-M and TASER-M Inclusion criteria -Confirmed HIV-1 infection; -≥18 years of age; Eligible a for initiation of a first-line ART regimen, or switch from a first-line ART regimen (containing at least 3 antiretroviral drugs and taken for at least 6 months) to a secondline ART regimen due to virologic, immunologic and/or clinical failure -Signed informed consent for study participation prior to enrolment Exclusion criteria Currently taking ART (minimum of 3 drug regimen), if initiating a first-line ART regimen b Pregnancy at enrolment c -HIV-1/2 dual-infection (in endemic countries only) c ART, combination antiretroviral therapy a Eligibility for ART initiation defined in accordance with national ART guidelines (i.e. advanced immunodeficiency as defined by CD4 cell count <200 or <350 cells/µl, or advanced clinical disease according to WHO clinical stage/CDC classification). b Specified PASER-M definition: re-initiation of a first-line ART regimen less than 30 days after stopping previous first-line ART (previous use of antiretroviral prophylaxis or mono/dual therapy is not an exclusion criterion). c Exclusion criteria applicable to PASER-M only F o r R e v i e w O n l y

F

  are n (%) of patients, unless otherwise indicated. ART, combination antiretroviral therapy; ARV, antiretroviral; CDC, US Center for Disease Control and Prevention; WHO, World Health Organization; PMTCT, prevention of mother-to-child transmission of HIV-1; TB, tuberculosis; IQR, interquartile range; NVP, nevirapine; d4T, stavudine; AZT, zidovudine; TDF, tenofovir; ABC, abacavir; 3TC, lamivudine; FTC, emtricitabine; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor a Eligibility for ART initiation in accordance with national ART guidelines (i.e. advanced immunodeficiency as defined by CD4 cell count <200 or <350 cells/µl, or advanced clinical disease according to WHO clinical stages or CDC classification). b Regimen switch due to treatment failure, defined by local standard of care guidelines as determined clinically, immunologically or virologically. c ARV-experienced is defined as any previous use of ARVs, i.e. (first-line) ART, mono/dual therapy, and/or PMTCT d Includes recipients of blood products, injecting drug users, perinatal transmission and unknown exposures

Table 1 (web appendix). Ethics approvals were obtained from the Academic Medical Center Institutional

	laboratory sites in 6 Asian countries (China, Indonesia, Malaysia, Philipines, South Korea,
	Thailand) are collaborating on TASER-M (Figure 1b). Ethics approvals were obtained from
	local IRBs having Federalwide Assurances (FWAs) in place from the United States Office for
	Human Research Protections. FWAs are required for TASER sites as they participate in the
	International Epidemiologic Databases to Evaluate AIDS (IeDEA) initiative, described
	elsewhere
	F o r
	R
	e v
	i e
	w
	O n l Review Board (IRB) and local IRBs. Sites are government, non-government, faith-based or y
	private clinics and hospitals, situated in major cities or rural areas. ART was introduced at the
	sites at varying time points between 1992 and 2006 (median: 2004). Eleven of 13 sites
	provide drugs, consultations and routine lab testing free of charge. HIV viral load testing is
	available at 8 of 13 sites.
	TASER collaborating sites are selected from within the existing TREAT Asia
	network based on their laboratory capacity to perform genotypic sequencing, described
	elsewhere (18, 19). Sites which do not have internal laboratory genotyping capacity can
	participate through collaboration with a TAQAS-certified laboratory. Eleven clinical and

Table 5 ,

 5 analysis of the first available 1 795 viral sequences demonstrated that the most common HIV subtypes in the cohort were C (1216, 68.7%), A (338, 18.7%) and D (179, 10.0%). The first PASER report published in 2008 reviewed the available data on

Table 1 .

 1 Patient eligibility criteria for PASER-M and TASER-M

Table 2 .

 2 Baseline patient characteristics, by network and line of ART

Table 3 .

 3 HIV subtypes and circulating recombinant forms by region and country

	Web Appendix

Table 1 .

 1 Characteristics of the PASER-M collaborating clinical sites (web appendix)

Table 2 .

 2 PASER and TASER genotyping laboratories (web appendix)

Table 4 .

 4 Baseline patient characteristics, by network and line of ART

				PASER-M (Africa)			TASER-M (Asia)
		Total	Initiation of first-line ART a	Switch to second-line ART b	Initiation of first-line ART a	Switch to second-line ART b
			ARV-naive	ARV-experienced c		ARV-naive	ARV-experienced c
	Patients -no. (%)	3713	2598 (87.0)	138 (4.6)	249 (8.3)	693 (95.2)	10 (1.4)	25 (3.4)
	Sex
	Female -no. (%)	1988 (53.5)	1494 (57.5)	105 (76.1)	124 (49.8)	239 (34.5)	10 (100.0)	16 (64.0)
	Male -no. (%)	1725 (46.5)	1104 (42.5)	33 (23.9)	125 (50.2)	454 (65.5)	0 (0.0)	9 (36.0)
	Age (years) -median (IQR)	36.9 (31.7-43.3)	37.0 (31.2-42.8)	34.7 (29.2-40.2)	38.6 (32.9-44.2)	36.5 (31.1-43.2)	33.1 (27.4-38.4)	36.5 (32.4-41.9)
	18-29	707 (19.0)	490 (18.9)	39 (28.3)	28 (11.2)	143 (20.6)	3 (30.0)	4 (16.0)
	30-39	1668 (44.9)	1176 (45.3)	65 (47.1)	112 (45.0)	298 (43.0)	5 (50.0)	12 (48.0)
	≥40	1338 (36.0)	932 (35.9)	34 (24.6)	109 (43.8)	252 (36.4)	2 (20.0)	9 (36.0)
	HIV exposure -no. (%)
	Heterosexual contact	2583 (69.6)	1731 (66.6)	108 (78.3)	191 (76.7)	520 (75.0)	10 (100.0)	23 (92.0)
	Homosexual contact	134 (3.6)	4 (0.2)	0 (0.0)	0 (0.0)	128 (18.5)	0 (0.0)	2 (8.0)
	Other d	996 (26.8)	863 (33.2)	30 (21.7)	58 (23.3)	45 (6.5)	0 (0.0)	0 (0.0)
	WHO clinical stage -no. (%)
	I	493 (16.5)	393 (15.1)	25 (18.1)	75 (30.1)	na	na	na
	II	711 (23.8)	623 (24.0)	33 (23.9)	55 (22.1)	na	na	na
	III	1281 (42.9)	1145 (44.1)	59 (42.8)	77 (30.9)	na	na	na
	IV	500 (16.7)	437 (16.8)	21 (15.2)	42 (16.9)	na	na	na
	CDC classification -no. (%)
	A	302 (41.5)	na	na	na	296 (42.7)	0 (0.0)	6 (24.0)
	B	166 (22.8)	na	na	na	152 (21.9)	10 (100.0)	4 (16.0)
	C	260 (35.7)	na	na	na	245 (35.4)	0 (0.0)	15 (60.0)
	Ever pulmonary TB -no. (%)	741 (20.0)	569 (21.9)	25 (18.1)	74 (29.7)	69 (10.0)	na	4 (16.0)
	Hepatitis B f -no. (%)	36 (4.9)	na	na	na	35 (5.1)	0 (0.0)	1 (4.0)
	Hepatitis C g -no. (%)	56 (7.7)	na	na	na	55 (7.9)	0 (0.0)	1 (4.0)
	History of ARV drug use -no. (%)	422 (11.4)	na	138 (100.0)	249 (100.0)	na	10 (100.0)	25 (100.0)
	ART	334 (9.0)	na	60 (43.5)	249 (100.0)	na	0 (0.0)	25 (100.0)

Table 5 .

 5 HIV subtypes and circulating recombinant forms a by region and country The collaborating sites in Philippines, South Korea and Indonesia had not yet provided sequence data at time of the current analysis.

	PASER-M (Africa)

a HIV subtypes were determined from the pol sequences, using the REGA HIV-1 subtyping algorithm version 2.0 (http://dbpartners.stanford.edu/RegaSubtyping) and/or Stanford HIV drug resistance database (http://hivdb.stanford.edu). b

Acknowledgements

The authors wish to thank the participants, site staff, and support staff at PharmAccess Foundation, Contract Laboratory Services, TREAT Asia and NCHECR. Special thanks to Donald Sutherland and Diane Bennett (World Health Organization) for encouraging the development of PASER and TASER.

Financial support

The PharmAccess African Studies to Evaluate Resistance is an initiative of PharmAccess Foundation, supported by the Ministry of Foreign Affairs of The Netherlands through a partnership with Stichting Aids Fonds (grant no. 12454). The TREAT Asia Studies to Evaluate Resistance is an initiative of TREAT Asia, a program of amfAR, The Foundation for AIDS Research, with major support provided by the Ministry of Foreign Affairs of The Netherlands through a partnership with Stichting Aids Fonds (grant no. 12454), and with additional support from amfAR and the National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health (NIH) and the National Cancer Institute (NCI) as part of the International Epidemiologic Databases to Evaluate AIDS (IeDEA) (grant no. U01AI069907). Queen Elizabeth Hospital and the Integrated Treatment Centre are supported by the Hong Kong Council for AIDS Trust Fund. The National Centre in HIV Epidemiology and Clinical Research is funded by the Australian Government Department of Health and Ageing, and is affiliated with the Faculty of Medicine, The University of New South Wales.

The funders had no role in the study design, data collection, data analysis, data interpretation, decision to publish, or writing of the report. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above.

Conflict of interest None declared