International Journal of Medicine and Medical Sciences ISSN: 2167-0404 Vol. 2 (11), pp. 221- 227, November, 2012. © International Scholars Journals

Case Report

Further validation of Zagazig depression scale shortened form (ZDS-SF) and depression diagnosis in a United Kingdom (UK) student population

Ahmed. K. Ibrahim^{1, 2}*, Shona. J. Kelly³ and Cris Glazebrook⁴

¹Public Health Division, Faculty of Medicine, Assiut University, Assiut, Egypt.

²Division of Epidemiology, Community Health Sciences School, D Floor, West Block

Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom (UK).

³Centre for Intergenerational Health Research, University of South Australia, Division of Health Sciences, Social Epidemiology Unit, City East Campus, Adelaide, Australia.

⁴Division of Psychiatry, Institute of Mental Health, Jubilee Campus, Nottingham, United Kingdom (UK).

*Corresponding author. E-mail: [email protected]

Received 09 July, 2012; Accepted 09 November, 2012

Abstract

The Zagazig Depression Scale has been validated in Egyptian populations and the shortened form (ZDS-SF) found high rates of depression in Egyptian students. Preliminary research has supported the validity and reliability of the measure in a UK student but further work is needed. The study aimed to determine the criterion validity of the ZDS-SF against a clinical interview and its test-retest reliability in a UK student sample. Participants (n=20) completed online measures of the Patient Health Questionnaire and the ZDS-SF at time 1. At time 2 (median follow-up 15 days) they were interviewed using the Clinical Assessment in Neuropsychiatry (SCAN) to establish a clinical diagnosis of depression, followed by re-administration of the online measures.  There was excellent (95%) agreement for diagnosis of depression between time 2 ZDS-SF and SCAN (Kappa = 0.89). The sensitivity of the ZDS-SF was 100% and the specificity 93.3%, giving an overall positive predictive value of 83.3%. The ZDS-SF symptom score also had good response stability over a two week interval (ICC = 0.66). ZDS-SF scale is a valid measure of depression for use in a UK university student cohort with good psychometric properties and can be used for cross-cultural comparison studies.

Key words: Depression, university student, Zagazig depression scale.

INTRODUCTION

Depression is recognized as the most common psychiatric disorder affecting adolescents and young adults   (Birmaher  et  al.,  2004).  It  has  a  multi-factorial

Abbreviations: ZDS-SF; Shortened form Zagazig depression scale, PHQ-9; patient health questionnaire-9, SCAN; schedule for clinical assessment of neuropsychiatry, DSM-IV; diagnostic and statistical manual of mental disorders, 4th edition, WHO; World Health Organization, ICD-10; International Classification of Disease, 10th revision, BDI; Beck depression inventory, SES; socio-economic status, GP; general practitioner, NHS; National Health Service, ICC; intra-class correlation, HDRS; Hamilton depression rating scale.

origin; hence many theories have been postulated to explain it (biological, genetic, environmental theories) (Hankin, 2006). Also, it is multi-faceted and can be presented by a mixture of psychiatric and/or physical symptoms (Weinberger et al., 2009). It is not an easy task to diagnose depression in adolescents and young adults as they report certain symptoms more frequently than adults (Gladstone et al., 2011). Although frequently a mild affective disorder depression can have serious complications in young adults and suicide is the leading cause of death in this age group (Mirjami et al., 2011). There is evidence that university students have high rates of depressive symptoms and early identification is important since depression is a preventable and treatable disease especially in children and young adults (Ibrahim et al., 2012; NICE, 2009).

The Arabic version of the Zagazig depression scale (ZDS) has been previously used to screen for depressive symptoms in the Egyptian general population (El-Sayeh, 1991; Fawzi et al., 1982; Fawzy, 1982; Shaheen and Fawzi, 1985) as well as in Egyptian university student cohort (Ibrahim et al., 2011a). It has been translated to be used as a tool for cross-cultural comparison between Egyptian and UK students and preliminary research has supported its validity and reliability in this population (Ibrahim et al., 2010). 

Few studies have explored test-retest reliability in depression scales in university students particularly. The beck depression inventory (BDI) multiple test-retest reliability was investigated in a university student sample over a two month period, concluding that BDI has a strong correlation over time (r = 0.9), however they did not look at the agreement with other scales (Ahava et al., 1998). This was further supported in another study, where the test-retest reliability was 0.96 over one month interval (Sprinkle et al., 2002). Test-retest reliability is considered good if the agreement between responses on separate administrations is high. Measures of stable constructs over time are expected to have high test-retest reliability. In contrast, since overall mood is anticipated to change over time, a mood scale with very high test-retest reliability may be less sensitive to mood changes (Valentin et al., 2002).

The interval between tests is therefore a key point to consider before interpreting the results of any test-retest analysis. Most depression scales ask participants about depression symptoms over the past two-four weeks. Administration of the depression scale after a longer interval might yield moderate test-retest reliability; because it is more likely that depression symptoms can change over

time. If administered two to four weeks apart, test-retest reliability should be moderate to high (William Li et al., 2010).

It was proposed that a standardized and validated tool for depression screening is necessary as it enables comparisons of findings both nationally and internationally and enhances the reputation of the measure (Laake et al., 2007). However, there is no universal agreement on how to adapt an instrument for use in another cultural setting (Gjersing et al., 2010). Self-reported depression scales are cost-effective in time and resources, especially in case of mass screening surveys (Jenkins and Dillman, 1995). Clinical interview is considered the gold standard for diagnosis of depression as it is more objective than subjective and it is carried out by well-trained personnel who can rate the symptoms accurately (Wing et al., 1990).

Aim of this research

To determine the criterion validity of the  ZDS-SF  against a clinical interview and the test-retest reliability of the ZDS-SF in a UK undergraduate student sample.

METHODOLOGY

Participants

Participants were university students who had taken part in an online survey of the socio-economic determinants of depression (Ibrahim et al., 2011b). Inclusion criteria were undergraduate students in completion of an online assessment of ZDS-SF and PHQ and attend either the University of Nottingham or Nottingham Trent University. Participants were excluded if they were EU or international students and if they did not provide contact details. Our minimum target sample size was 20 students which was sufficient to detect a Kappa of 0.7 with 90% power based on an estimate of 20% positive ratings (Sim and Wright, 2005).

Of the 923 students participating in the online survey, 564 met the inclusion criteria and were invited to take part in further study. A total of 184 students (32.6%) supplied contact details of which 96 met the inclusion criteria and were invited to participate in the validation study. Of the 34 who agreed to be interviewed 14 either failed to make an appointment or failed to attend for interview within the study period, giving a final sample of 20.

Study design

The validation study consisted of a cross-sectional standardized face to face psychiatric interview with repeated measures for ZDS-SF scores. 

Measures

Zagazig depression scale shortened form (ZDS-SF)

The ZDS was derived from the Hamilton depression scale (HDRS) (Hamilton, 1967). The original Arabic version contained 52 items representing 17 domains ((Fawzi et al., 1982). In the current study the translated, modified 43-item version of the ZDS was used (Ibrahim et al., 2010, 2011a), which consists of 11 domains (depressed feelings, suicide ideation, guilt feelings, anxiety, insomnia, agitation/hypochondriasis, sleep maintenance, diminished ability to think, concentrate or slowness, lack of energy and motivation, weight loss, sexual symptoms). Participants rate symptoms as present (0) or absent (1) during the last two weeks giving a maximum score of 43. The reliability and validity of the ZDS-SF was tested in two studies (Ibrahim et al., 2010, 2011a). The first was a pilot study that used a  sample  of 275 UK University students to improve the questionnaire wording and layout. This pilot study (estimated 30 to 40% response rate) found a strong (r = 0.8) and statistically significant correlation between the Patient Health Questionnaire-9 (PHQ-9) (Spitzer et al., 1999) which has been well validated for use as a depression screening tool in UK adult population (Lowe et al., 2004; Spitzer et al., 2004). Internal consistency for the total ZDS-SF was excellent (Cronbach's alpha = 0.90) (Ibrahim et al., 2010). The second was a reanalysis of a representative sample of Egyptian university students, revealed that the internal consistency of the revised ZDS-SF was excellent (Cronbach's alpha = 0.91) as was the spilt-half correlation coefficient (r = 0.81, p < 0.001) (Ibrahim et al., 2011a). We used a cut-off of 10 proposed by the ZDS developers (Fawzi et al., 1982) to categorize participants as depressed or non-depressed. ZDS-SF scores were also used as a continuous variable in some analyses.

Patient health questionnaire-9 (PHQ-9)

PHQ-9 is the depression module of the PHQ (Spitzer et al., 1999). It is composed of 9 items each representing one of the 9 DSM-IV criteria for depression. It uses a 4-point scale; "not at all", "several days", "more than half the days" or "nearly every day". The maximum possible score for the PHQ-9 is 27, with a cutoff of 5 to indicate the presence of at least mild depression (Spitzer et al., 1999). The validity, feasibility, and ability to detect changes in depressive symptoms have been reported in several studies (Kroenke et al., 2001; Liu et al., 2011; Lowe et al., 2004; Spitzer et al., 1999; Wulsin et al., 2002). Additionally, the PHQ-9 is increasingly being used in research, and has demonstrated superior criterion validity with respect to the diagnosis of depression compared with other established screening instruments for depression (Kroenke et al., 2004; Lowe et al., 2004).

Schedule for clinical assessment in neuropsychiatry (SCAN)

Schedule for clinical assessment in neuropsychiatry (SCAN) is a set of instruments and manuals designed to assess, measure and classify psychopathology and behavior associated with major psychiatric disorders in adults. Developed under the aegis of the World Health Organization (WHO), it has a bottom-up approach where no diagnosis-driven frames are used to group the symptoms but rather each symptom is assessed in its own right. It has a proven stability and robustness to differentially assess psychotic and neurotic states (Wing et al., 1998). The validity, reliability and the psychometric prosperities of the SCAN to detect changes in a wide variety of neuropsychiatric disorders have been supported    in      several      studies      (Forsell,      2005; Krisanaprakornkit et al., 2006, 2007; Piyavhatkul et al., 2008; Schutzwohl et al., 2007). To assess the subjects the interviewer conducts a semi-structured, standardized clinical interview. The order in which the sections are completed depends on the most important symptoms of the respondent. This lack of a fixed order makes it very flexible and versatile. Rating is done on the basis of matching the answers of the respondent against the definitions of the symptoms in the SCAN glossary (Wing et al., 1998). In the current study we used the depression section (6th section) in the manual. After completing the interview the data were entered into the laptop version of SCAN "Ishell". Subsequently the data were fed into algorithms for ICD-10 and DSM-IV diagnoses. These algorithms produce a diagnostic classification for depression and a list of symptoms. The severity of the condition is classified as mild, moderate or severe.

Procedures

At time 1 participants completed online versions of the ZDS-SF and PHQ sequentially and, together with demographic details. Participants meeting the study inclusion criteria and agreeing to follow-up clinical interview were offered a choice of interview dates (median follow-up 15 days). The diagnostic interviews (SCAN) were administered at time 2 in a quiet studio. Interviews ranged between 40 to 80 min in duration, with an average of 60 min. All diagnostic interviews were administered by the same trained and reliable clinician (AKI) who was blind to participants’ time 1 ZDS scores. Immediately after the SCAN interview, participants were asked to complete the online version of ZDS-SF followed by the online PHQ.

Statistical analysis

A Kappa analysis (Cohen, 1960) was conducted to explore the degree of agreement between the ZDS-SF and PHQ and SCAN (concurrent validity). According to Fleiss a kappa over 0.75 is considered as excellent, 0.40 to 0.75 as fair to good, and below 0.40 as poor (Fleiss, 1981). Sensitivity and specificity and other validity measures were also calculated. All analyses were carried out using STATA version 10.1 software (STATA, 2008).

Ethical considerations

This study received approval from the Medical School Ethics Committee of Nottingham University

Ref. No. N/9/2008 as a compensation each student participated in was offered a £10 gift voucher for a local department store. A signed written consent was obtained before starting the interview. If the student was

Table 1. Description of the interviewed students.

diagnosed by SCAN as depressed, an e-mail was sent directing the participant to contact his or her GP, NHS direct, the University Counseling Services or the researcher to make the necessary arrangements.

RESULTS

Description of the interviewed sample

Detailed socio-demographic characteristics of the sample are shown in Table 1. Males and females were equally represented in the sample and there was a reasonable range in terms of socio-economic background. The age of the students sampled ranged from 18 to 34 with a mean (SD) of 20.7 years (6.9). There was an equal distribution in the mother’s educational levels, but fathers were more likely to have higher education. Additionally, parental occupational distribution was more or less equal. As expected the majority of students (60%) were in the high affluent group as measured by the family affluence scale (Boyce et al., 2006) (Table 1).

SCAN diagnoses of the 20 interviewees

The prevalence of psychiatric disorders in the current sample at time 2 as ascertained by the clinical interview

was 40% (95% CI; 38.9-40.4) (n=8). The most common SCAN diagnosis in the interviewed students was depressive disorder (n=5), alcohol dependence (n=2), anxiety and obsessive compulsive disorders (n=1). One student had minor depressive disorder and alcohol abuse Table 2 shows the detailed description of the 20 interviewed students regarding their SCAN diagnosis, socio-demographic characters and their ZDS and PHQ-9 scores. 

ZDS-SF reliability and validity

Depression scores

The mean ZDS-SF score for the 20 participants at time 1 was 11.80, (SD = 4.3). The 5% trimmed mean was 11.17, only 0.63 below the sample mean. Scores were essentially normally distributed (Skewness (SE) = 0.82 (0.36), Kurtosis (SE) = 0.94 (0.57)); with a median score of 8.5. Five participants (25%) scored above the cut-off for depression on the ZDS-SF at time 1, all of whom were later classified as cases of depression by the SCAN clinical interview at two-week follow-up (100% agreement, Kappa = 1). At time 2 the mean ZDS-SF score was 14.62, (SD = 4.5). The 5% trimmed mean was 14.36, only 0.26 below the sample mean. Scores were also normally distributed (Skewness (SE) = 0.69 (0.23), Kurtosis (SE) = 0.72 (0.47)); with a median score of 15. Of the 20 participants, six (30%) scored above the cut-off for depression on the ZDS-SF at time 2. A quarter of the total sample (n=5) was diagnosed as depressed using the SCAN diagnostic classification. Four were classified as having mild depression and one was classified as moderate depression.

Concurrent validity

There was a strong positive correlations between ZDS-SF scores at time 2 and SCAN diagnostic symptom scores (Spearman’s Rho = 0.88, p < 0.001). There was agreement between ZDS-SF and SCAN on whether the participant was depressed or not depressed for 95% of cases. In only one case (5%) participants were classified as depressed by the ZDS-SF (symptom score = 18) and not by the SCAN. The resulting Kappa score (0.89) indicates excellent agreement (p < 0.001) (Table 3). The sensitivity of the ZDS-SF was 100% and the specificity was 93.3% giving an overall positive predictive value of 83.3%. For the PHQ there was a moderate positive correlation between PHQ scores at time 2 and SCAN diagnostic results (Spearman’s rho = 0.54, p < 0.001) with lower sensitivity and specificity (Table 4).

Reliability

The time interval between the test and the retest ranged from 10 to 30 days with a mean of 17 ± 5.5 days (median

*ZDS-SF cutoff ≥ 10, PHQ cutoff ≥ 5.

Table 4. Other validity measures for ZDS-SF vs. PHQ and SCAN.

15 days, IQR range 12 to 27 days). The 43-item version ZDS had good response stability over about two weeks interval (n=20, Spearman’s correlation = 0.72; intra-class correlation coefficient (ICC) = 0.66 (95% CI=0.58-0.69). Depression scores increased over time from a mean of 11.8 (± 4.3) at time one to 14.6 (± 4.5) at time two, (t = 3.7, df = 38, p < 0.001). The internal consistency of the ZDS-SF was very good for both test and retest (Cronbach’s alphas 0.88 and 0.89 respectively).

International Journal of Medical and Medical Sciences ISSN: 2167-0404 Vol. 2 (11), pp. 218-220, November, 2012.  © International Scholars Journals

Case Report

Case report of non-convulsive status epilepticu

R. Stephen Griffith¹, Chad Sharky¹ and Angela Divjak

Community and Family Medicine, University of Missouri-Kansas City School of Medicine, 7900 Lee’s Summit Road, Kansas City, MO  64139, USA.

*Corresponding author. E-mail: [email protected]

Received 13 June, 2012; Accepted 16 November, 2012

Abstract

Non-convulsive status epilepticus (NCSE), like the easily recognized convulsive status epilepticus, is a condition requiring prompt treatment. However it is often unrecognized as the cause of mental status impairment or coma. Herein we describe a case in which treatment of a patient was delayed due to the lack of recognition of NCSE. A brief review of NCSE follows, including its presentation, diagnosis, and treatment. Clinicians should be aware of NCSE and include it in the differential diagnosis when treating a patient with unexplained mental status change.

Key words: Non-convulsive status epilepticus, mental status, coma.

INTRODUCTION

Status epilepticus (SE) with generalized convulsions is an easily recognized medical emergency well known to requiring prompt treatment to prevent morbidity and mortality. However, in the absence of obvious convulsions, SE may not be recognized. Non-convulsive status epilepticus (NCSE) is often unrecognized as the cause of mental status change or coma, and thus treatment is delayed (Meierkord and Holtkamp, 2007). NCSE can have various presentations that can be related to mental status (confusion, unresponsiveness, psychosis, coma), motor symptoms (unusual position of limbs or head, myoclonias, twitches), and/or automatisms (verbal, mimicry) (Chang and Shlomo, 2011). Lacking the typical tonic-clonic movements of SE, the diagnosis is often missed. The outcomes of patients experiencing NCSE range from benign to fatal, related to the cause. Therefore it is of critical importance that NCSE be recognized and managed in a timely manner (Meierkord and Holtkamp, 2007). Here we describe such a case in a woman with no previously known seizure history and in whom the diagnosis was delayed over 18 h. Following the case presentation, a brief review of NCSE is provided.

CASE PRESENTATION

KSB is a 43 year old female brought to the emergency department (ED) by ambulance after her mother discovered her at her home unable to respond to simple commands, though awake. There were no signs of trauma. Emergency responders noted she was able to make eye contact when addressed, but was not responsive in any other way. Upon arrival in the ED, her fingerstick glucose was found to be <69 mg/dL, so dextrose was given intravenously, with no effect. A fluid bolus of 1 L of normal saline was also given. Initial vital signs were: T-97; BP-154/93; P-63; RR-14; oxygen saturation 99% on room air. Physical examination was unremarkable save the mental status changes.  CT scan of the brain was normal. The patient was admitted to the ICU for monitoring.

The past medical history (provided by her mother) was remarkable for anorexia nervosa, hepatitis C, anxiety, attention deficit disorder of adulthood, gastroesophageal reflux disease, and “hepatic encephalopathy.” She had undergone an appendectomy in the past. Current medications included: hydroxyzine, 25 mg four times a day, clonazepam 2 mg four times a day, buproprion SR, 300 mg each morning, 150 mg at night, metoclopramide 10 mg before meals and at bedtime, eszopiclone, 3 mg at bedtime, fluoxetine 20 mg daily, ranitidine 150 mg twice daily, docusate 100 mg twice daily, baclofen 10 mg three times a day as needed, lactulose in uncertain dose, hydrocodone three times a day as needed, and amphetamine and dextroamphetamine in uncertain dose. There was no contributory family history. Social history revealed the patient was unemployed, living with her mother and daughter, with a previous history of multiple drug abuse (no recent history), occasional binge drinking, and smoking ½ packs per day for over 20 years. Her exam in the ICU revealed a nonresponsive female. While awake and alert, eye contact was infrequent and there was no response to verbal cues. Neurologic examination was limited due to unresponsiveness. Pupils were 5 mm and reactive. Ocular vestibular reflexes were intact and deep tendon reflexes were normal. The laboratory examination included a normal complete blood count and urinalysis. There were no detectable salicylates or acetaminophen. Her chemistries were normal other than sodium of 131 mmol/L, a chloride of 96 mmol/L, and a glucose of 226 mg/dL after the glucose infusion. Her ammonia level was 22 umol/L (normal 16 to 60). Her urine drug screen was positive for benzodiazepines, but negative for barbiturates, amphetamines, cannabinoids, cocaine, opiates, and phencyclidine. Her alcohol level was 8 mg/dL.

A neurological consultation documented myoclonic twitching movements of the torso and upper extremities. At that time the diagnosis of non-convulsive status epilepticus was considered.  Intravenous lorazepam was given with a rapid improvement in the patient’s mental status. After giving her usual dose of clonazepam, the patient’s mental status was normal. A few hours later she insisted on leaving the hospital. The patient failed to keep her appointment for an EEG.

DISCUSSION

NCSE is a heterogeneous clinical disorder broadly defined as prolonged seizure activity in the absence of major motor signs. As such, the incidence is difficult to assess. It is estimated at 2 to 8 per 100,000 per year with a marked age association—in geriatric population’s estimates of 55 to 86 per 10,000 per year have been reported (Meierkord and Holtkamp, 2007). It may comprise up to 1/3 of all patients with SE (Walker, 2007). Of note is that 58% of NCSE diagnoses occurred in people with no previous history of a seizure disorder (Chang and Shlomo, 2011). In comatose patients, 8% without clinical signs of seizure activity were found with EEG to be in NCSE (Towne et al., 2000).

The clinical presentation of NCSE is typically understated. In a retrospective review of 23 patients with NCSE presenting to the emergency department with altered mental status, only 13 were diagnosed in less than 24 h (Kaplan, 1996). The three major clues to the diagnosis of NCSE include an abrupt onset, fluctuating mental status, and subtle clinical signs such as eye fluttering and various automatisms (involuntary, autonomic movements that occur with an alteration in consciousness, including those that are gestural (for example, picking movement with the fingers) or oroalimentary, for example, lip smacking or stuttering) (Chang and Shlomo, 2011; Towne et al., 2000; Kaplan, 1996; Kaplan and Stagg,  2011).  Mental  status  changes can be on a spectrum from slow mentation and responses to confusion or psychosis to complete unresponsiveness (Wells et al., 1992). While there are no universally agreed upon diagnostic criteria (Meierkord and Holtkamp, 2007), and no pathognomonic EEG changes in NCSE, persistent abnormalities without convulsive activity are the “gold standard” of diagnosis (Chang and Shlomo, 2011). For those patients with an unexplained changes in behavior or mental status, with features highly suggestive of NCSE such as eye movement abnormalities, aphasia with a history of absence seizures, or with risk factors for seizures such as stroke, neoplasia, dementia, history of demyelinating syndromes (that is, multiple sclerosis), or previous neurosurgery, an EEG is clearly indicated (Meierkord and Holtkamp, 2007; Wells et al., 1992; Primavera et al., 1996; Hussain et al., 2003). A therapeutic trial of a benzodiazepine may be diagnostic if an EEG is not available, but may confuse further diagnostic evaluation due to sedation.

NCSE may be subdivided into clinical forms; however, there is no general agreement on the various types. One straightforward categorization suggests three types: 

1. Absence—characterized by sudden onset of unresponsiveness with fluctuating lethargy, disorientation, or slow speech, and subtle clinical signs such as automatisms.

2. Complex partial—associated with impaired consciousness, a cessation of verbal and motor activities, and may have an aura prior. (Aphasic NCSE is a sub-type of complex partial NSCE, in which the sole manifestation of SE is aphasia, with reports of SE lasting up to 21 days before a diagnosis was discovered (Wells et al., 1992).

3. Subtle generalized—a natural progression of untreated or insufficiently treated generalized tonic-clonic SE in which motor phenomena are exhausted with motor activity greatly diminished. Patients are usually stuporous/comatose (Chang and Shlomo, 2011).

NCSE can occur de novo or develop after a convulsive event. It should be included in the differential diagnosis of any patient with new-onset altered behavior of undetermined cause. It should be especially considered in patients who fail to awaken after a generalized seizure (Chang and Shlomo, 2011). Some of the other diagnoses that can mimic NCSE are listed in Table 1 (Meierkord and Holtkamp, 2007). There are many potential etiologies including metabolic causes, neoplasms, infections, drugs, and toxins. Table 2 lists some reported causes (Maganti et al., 2008).

The clinical consequences of NCSE vary according to the cause and the time to recognition and treatment. In patients with pre-existing epilepsy, the prognosis is better than if the cause is related to an acute neurological or systemic disorder (Meierkord and Holtkamp, 2007).

Table 1. Disorders mimicking non-convulsive status epilepticus.

*From Meierkord and Holtkamp (2011). 

Table 2. Conditions associated with non-convulsive status epilepticus.

*Adapted from Maganti et al. (2008); **Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke.

 Animal studies indicate the potential for neuronal damage exists; however available human data indicate most clinical forms of NCSE are benign in terms of morbidity and mortality. Subtle generalized seizures which are more often due to an underlying systemic cause, such as post-hypoxic or septic  encephalopathies  are  associated with a high mortality rate, (Holtkamp and Meierkord, 2011)  especially in elderly, critically ill individuals (>50%) (Meierkord and Holtkamp, 2007).

Quantitative estimation of toxoplasma B1gene using real time polymerase chain reaction (PCR) in infected symptomatic and asymptomatic clinical cases 

Amany A. Abd El-Aal¹*, Maysa M Kamel¹, Eman y shoeib¹, Fawzia A Habib², Manal Barakat³, Lamia A. El-Housseiny⁴

¹Department of Parasitology, Faculty of Medicine, Cairo University, Egypt.

²Department of Obstetrics and Gynecology, Faculty of Medicine, Taibah University, Saudi Arabia.

³ Department of Clinical Pathology, Faculty of Medicine, Cairo University Egypt.

⁴ Department of Microbial Biotechnology, National Research Centre, Giza, Egypt.

*Corresponding author. E-mail: [email protected]  

Received 21 July, 2012; Accepted 29 November, 2012

Abstract

Serological diagnosis of active Toxoplasma infection is unreliable because reactivation of latent, hidden infection is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Data concerning the relation between the severity of infection and the parasitic load especially at human level is limited. A trial was done in this work to study the relationship between the virulence of the parasite in different clinical forms and parasitic genomic load, via SYBR® Green quantitative PCR amplification assay and primers targeting Toxoplasma B1 gene.  Fluorescence signals were generated from 91 samples, which were as follow; 7 cases with congenital manifestations, 19 cases with neurological disorders, and 65 asymptomatic cases.  The seven congenitally infected cases showed significantly higher parasite load (5.15x 10⁸  to 9x 10¹⁰). Other clinical forms showed genomic concentration ranged from 1.7x 10⁴ to 4.1x 10⁸. Equal copy numbers of template did not produce equal parameters on derivative melting curve plots. In conclusion, quantitative polymerase chain reaction (PCR) provides a sensitive and practical method not only for gene quantification, but also for gene scanning by melting curve analysis which is highly recommended for different Toxoplasma genes especially those with known pathological functions.

Keywords: Toxoplasma- Real time polymerase chain reaction (PCR)-B1 gene-clinical forms.

INTRODUCTION

Toxoplasma gondii (T. gondii) is an intracellular parasite, has very low host specificity, and it will probably infect almost all mammal and birds (Adriana et al., 2006). It is endemic worldwide and 15 to 85% of the human populations are asymptomatically infected.  Most cases of human infection are mild, but devastating disease can occur in immune compromised individuals and congenitally infected fetuses in which serious neurological or   ocular   problems  that   appear   either early after labour or later on during life and may not become manifested until the second or third decade of life. The progression and severity of the disease differ in patients due to several variables, including host and parasite genetics (Flegr, 2007). Prospective and retrospective studies of infants with subclinical toxoplasmosis at birth have revealed late onset squeals, including visual and hearing deficits, neurological deficits (such as seizures and micro-encephaly) and low IQ scores (Wilson et al., 1999; Jeffery, 2003). Schizophrenia is also reported to be correlated with Toxoplasma infection by Flegr et al. (2007).

Serological diagnosis of active infection is unreliable because reactivation of hidden infection is not always accompanied by changes in antibody levels, and evolution of a latent or dormant toxoplasmosis is highly unpredictable (Flegr, 2007). Moreover, reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection (Reischl et al., 2003). Application of polymerase chain reaction (PCR) has evolved as a sensitive, specific, and rapid method for the detection of T. gondii Deoxyribonucleic acid (DNA) in different samples, accordingly serving confirmation of active infection (Yang et al., 2009). Accelerating the molecular diagnosis of toxoplasmosis by performing quantitative real time PCR (qPCR) protocols has been reported (Lin et al., 2000; Costa et al., 2001; Bell and Cartwright, 2002; Reischl et al.,  2003; Contini et al., 2005; Adriana, 2006; Mesquita et al., 2010; Pignanelli, 2011). However, data concerning the relation between the severity of infection and the parasitic load especially at human level is limited.

The aim of the present work was to study the relation between the severity of Toxoplasma infection and parasite genomic concentration of recently infected symptomatic and asymptomatic clinical cases, via SYBR® Green quantitative PCR amplification assay

 METHODOLOGY

Sample collection and ethical issue

DNA templates extracted from blood of 92 positive Toxoplasma samples were kindly provided by Dr. Fawzia habib, Vice Dean of Taibah University, Saudi Arabia. These cases were serologically IgG negative and were diagnosed by detection of Toxoplasma B1 gene using a nested–PCR.

The extracted DNA samples were stored at –70°C until used. These samples were collected from patients attending the outpatient clinic of Taibah University as well as governmental hospitals in Almadinah, Saudi Arabia the period from January 2007 to February 2010 and supported by 2 projects.

These projects (number 27 to 33 and 421 to 430) were funded by King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia and Deanship of scientific researches, Taibah University, Kingdom of Saudi Arabia respectively. In this project, there was a collaboration with many Egyptian researchers, including our team, therefore, they supplied us with these positive samples. According to the clinical data of these Toxoplasma positive cases were as follow; seven cases of severe clinical forms of toxoplasmosis in the form of congenital anomalies, abortion or still birth, 19 cases with neurological disorders of unknown etiology (8 adults and 11 children) and 65 asymptomatic cases (diagnose during pregnancy).

Quantitative real time polymerase chain reaction (PCR) protocol

qPCR was performed with the LightCycler^® fastStart DNA Master SYBR Green dye, using the LightCycler^® 1.x/ 2.0/ 480 instrument (Roche Diagnostics, Hoffmann-La Roche Ltd, USA). Primers from bases (5´-CCG TTG GTT CCG CCT CCT TC-3´) and (5´-GCA AAA CAG CGG CAG CGT CT-3´) were used to amplify Toxoplasma B1 gene of 35-fold repeats. The resulting PCR fragment of T. gondii was analyzed using the LightCycler® Red 640 (detected in channel 640). The supplied standard row was used to determine the linear range of the reaction and to estimate the quantity of the target sequence in unknown samples. Software data analysis version 3.5.3 was applied as described in the LightCycler^® instrument operator's manual. The reaction mixture (20 µl; Master SYBR Green kit; Roche Diagnostic) contained 0.5 µM of each primer, 5 mM MgCl₂ and 5 µl templete DNA. All capillaries were sealed, centrifuged at 500 g for 5s, and then amplified in a LightCycler instrument. Amplification was performed for 50 cycles: 5 s denaturation at 95°C, 10 s annealing at 61°C and 15 s extensions at 72°C, with an overall ramp rate of 20°C s. A single fluorescence reading for each sample was taken at the extension step. Quantitative results were expressed by determination of the detection threshold or the crossing point (Cp), which marked the cycle when the fluorescence of the given sample significantly exceeded the baseline signal. They were expressed as a fractional cycle number. Then, the crossing points (Cps) were plotted against the known parasite concentration to obtain a standard curve. The parasite count for a given sample was calculated by extrapolation from this standard curve (positive control). Positive sample specificity was confirmed by determining the melting curve with different values of melting temperatures (T_m) (95°C, 4.40°C/s ramp rate; 40°C, 2.20°C/s ramp rate; 65°C, 4.40°C/s ramp rate, 95°C, 0.02°C/s ramp rate continuous measurement). All data concerning Cps, melting curve parameters of the amplified products were calculated. Statistical analysis was done using the SPSS program version 0.13. ANOVA followed by post Hock test were applied and interpreted at the 5% level of significance.

Positive and negative control

A standard row was generated using the provided cloned and purified Toxoplasma DNA (Roche Diagnostics), allows for the absolute quantification of the unknown samples. The standard was prepared with concentration in the range from 10⁶ copies/rxn to 10 copies/rxn of T. gondii and the CPs calculated with second derivative

Table 1. Range of Toxoplasma genomic concentrations and crossing point in different clinical forms of Toxoplasma diseased cases.

maximum method. To prepare the standard row: 6 different quantities provided with the kits to yield 10 to 10⁶ target molecules in 5µl once resuspended. A hole through the sealing foil was punched. 45 µl PCR- grade water was added to each vial of the row. The target DNA was mixed by pipetting the solution up and down 10 times. Negative control: By replacing the template DNA with water. PCR product was obtained within one hour.

RESULTS

Fluorescence signals were generated from all samples when analyzed by SYBR® Green qPCR except one sample. Test for inhibitors was not done; therefore, the negative sample was excluded during further analysis. Quantitative genomic estimation of these positive samples in qPCR was ranging from 1.7x 10¹ to 9x 10¹⁰. The symptomatic infected Toxoplasma cases (7 congenitally infected and 19 with neurological manifestation) showed significant higher parasitic load (P< 0.05) (from 5.15x 10⁸  to 9x 10¹⁰) than those with asymptomatic infection in which parasitic load was ranging from 1.7x 10¹ to 1.5x 10⁸ (Table 1). No significant difference was noticed between the symptomatic groups (P> 0.05). Crossing points (Cps) showed different values ranging from 39.68 to 12.88 reflecting the different DNA quantities (from 1.7x 10¹ to 9x 10¹⁰) respectively (Table 1). Applying gene scanning option and high resolution melting curve analysis, the software of the programmed LightCycler^® 1.x/ 2.0/ 480 instrument category, positive samples in different groups showed different values of T_m that were varied from 84.2 to 87.07 (Figure 1). The means of T_m were estimated to be 85.6362 ± 0.57950 SD, 85.7467± .44802 SD and 85.8640 ± 0.68857 SD. The maximum and minimum values in the different groups were 84.20 to 86.76, 85.27 to 86.49 and 85.37 to 87.07 respectively. The ranges of different melting curve parameters (area, width and heights) were 2.56 to 67.14, 3.16 to 4.92, and 0.73 to 15.4 respectively. Equal copy numbers of template did not produce equal parameters on derivative melting curve plots for example, samples obtained concentration around 10⁷ showed different heights ranging from 12.5 to 2.8. There was significant difference concerning areas of melting curve (P < 0.05). Otherwise, no significant correlation was noticed between parasite load and different T_m parameters (area width and height), (P> 0.05).   

DISCUSSION

T. gondii is an ubiquitous parasite found in all classes of warm-blooded vertebrates. Nearly one-third of humans have been exposed to this parasite (Yang et al., 2009).  Factors that regulate the virulence and pathogenesis of Toxoplasma are still poorly understood. When acquired during pregnancy, toxoplasmosis can be disastrous, leading to fetal loss or conversely to subclinical disease. In congenitally infected infants, evolution is highly unpredictable (Peyron et al., 2004). These increase the demand of quantitative assays to monitor the severity of the parasitic infection in different clinical situations.  Recently, assays targeted LightCycler instruments have been greatly developed and provided with variable software technologies which expand the performance on different optional analytic parameters.

Unfortunately, no commercially available PCR kits for routine diagnosis of toxoplasmosis, but only limited for research purposes. Even so, we could not start working in this study, except after receiving positive Toxoplasma samples from  our colleagues in Saudi Arabia who have finished a funded project in which hundreds of human blood samples were screened for the presence of  Toxoplasma B1 gene using nested PCR. In general there is limited data concerning the relation between the intensity of infection and the parasitic load especially at human level. A trial was done in this work to study the relationship between the severity of infection and parasitic load in blood applying SYBR® Green quantitative PCR amplification assay.

The present study included 91 cases ranging between asymptomatic and sever Toxoplasma infection that were previously diagnose by nPCR. Quantitative Toxoplasma genomic estimation was performed on LightCycler^® 1.x/ 2.0/ Roche 480 instruments with a SYBR Green

technique. Negative control did not generate any fluorescence signal, while positive samples showed signals at different crossing points (CPs) depending on the number of genomic materials which were  ranging  from 10¹ to 10¹⁰  reflecting different parasite load. The severely manifested,  congenitally infected (7 cases) showed significantly higher DNA concentrations (from 5.15x 10⁸ to 9x 10¹⁰) and other clinical forms showed different concentrations of the genomic materials (from 1.7x 10¹ to 4.1x 10⁸).  Therefore, high parasite load does not always progress to active infection that resulting in serious illness. This was explained by some authors being as a result of different genotypes of Toxoplasma parasite that lead to different clinical forms of the disease (Howe et al., 1997; Fuentes et al., 2001; Saeij et al., 2005).

International Journal of Medicine and Medical Sciences ISSN: 2167-0404 Vol. 2 (11), pp. 232-235, November, 2012. © International Scholars Journals
 

Full Length Research Paper

The association of ABO blood group and urinary schistosomiasis in the Middle Awash Valley, Ethiopia

Ketema Deribew¹ _, Zinaye Tekeste ²* and Beyene Petros³

¹Department of Biology, Bonga Teacher Education College, P. O. Box 91, Bonga, Ethiopia.

²School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, Gondar University,

P. O. Box 196, Gondar, Ethiopia.

³Department of Microbial, Cellular and Molecular Biology, College of Natural Sciences, Addis Ababa University,

P. O. Box 1176, Addis Ababa, Ethiopia.

*Corresponding author. E-mail: [email protected]  

Received 08 July, 2012; Accepted 21 November, 2012

Abstract

The present study was carried out in the Middle Awash Valley, Ethiopia to determine if there was any relationship between the blood group of the human host and urinary schistosomiasis. Patients and Urine and blood samples were collected from 370 children (95 infected and 275 healthy controls) aged 5 to 15 to examine urinary schistosomiasis, hemoglobin concentration and to type blood groups. There were 23 (47.9%) blood group A, 11 (22.9%) blood group B and 2 (4.2%) blood group AB children in the severe schistosomiasis category. Blood group O made up only 25% of severe schistosomisis patients compared to 56.3 and 53.1% of the mild and healthy controls, respectively. A significant difference was observed in egg load between children with blood group O, 1.63 (0.39) (mean ± SD) eggs/10 ml urine) and A,1.93 (0.45) eggs/10 ml urine (P = 0.03). Although the mean egg load in children with blood group O was higher than in those with blood group AB, 1.45 (0.21) egg/10 ml urine), the difference was not significant (P = 0.54). As compared to the mild schistosomiasis cases, the case of severe schistosomiasis was more likely to be of type A (SS vs. MS: O vs. A, odds ratio (OR) 0.23, 95% confidence interval (CI) 1.62-11.42) and B (SS vs. MS: O vs. B, OR 0.24, 95% CI 1.24-13.76) than type O. The study showed that on the basis of egg load, used to determine severity of urinary schistosomiasis, children with blood group A and B were highly prone to severe urinary schistosomiasis as compared to children with blood group O.

Key words: ABO blood group, urinary schistosomiasis, egg load, mild schistosomiasis, severe schistosomiasis. 

INTRODUCTION 

Schistosomiasis or biharzia is a tropical disease caused by blood-dwelling fluke worms of the genus Schistosoma. Most infections in human caused by Schistosoma haematobium, S. japomicum and S. mansoni, together with a minor contribution from S. intercalcatum and S. mekongi.

Schistosomiasis is estimated to infect 200 million people around the world while about 600 million are   estimated   to   be   at   risk   of   infection  (World  Health Organization, 1998). In Ethiopia, S.haematobium is endemic in the awash valley (middle and lower awash valley) from Dahitele irrigation scheme on the Amibara/Angele plain to Gewane, Dubti and Assaita area. In the Wabishebele valley (lower Wabi valley), near the Somali border in the villages of Kelafo, Mustahil and Burukur the parasite is reported (Birrie et al., 1998). It is also reported from the western lowlands of Ethiopia such as Kurmuk (Birre et al., 1996).

The relationship between ABO blood groups and susceptibility, resistance, or severity of some diseases has been studied and close correlations demonstrated between blood groups and malaria  (Zinaye  and  Beyene 2010), S. mansoni (Ndamba et al., 1997) and oesophagogastric varices (Amer et al., 1971). However, the effect of the ABO blood group on urinary schistosomiasis has received little attention.

However, some studies reported absence of significant association between urinary schistosomiasis and ABO blood groups (Ndamba et al., 1997; Kassim and Ejezie, 1982; Khattab et al., 1968).

For instance, Kassim and Ejezie (1982) reported no significant association between the ABO blood group and S. haematobium from two hundred and sixty nine individuals in Epe, Nigeria. In contrast, Ndamba et al. (1997), reported that intensity and annual incidence of S. haematobium infection and related organ pathology was significantly higher among children of blood group A and lowest among blood group O children.

These contradictory reports on the association of ABO blood group and urinary schistosomiasis shows the complexity of the interaction between the parasite and the host.

Therefore, the present study was carried out in the Middle Awash Valley, Ethiopia to generate additional data on the association between the blood group of the human host and urinary schistosomiasis.

 MATERIALS AND METHODS

 Study area and population

 The study was conducted in the Middle Awash Valley, Ethiopia. The area is known for long to be infested by the snail intermediate host, Bulinus abysinicus of S. haematobium. The study participants were Afar ethnic group.  Middle Awash is located at 290 km to the east of Addis Ababa and it is endemic for urinary schistosomiasis (Kloos et al., 1978).

A total of 370 children (95 S. hematobium positive and 275 healthy controls) aged 5 to 15 participated in the study.

Of the total 95 S. hematobium patients, those harboring > 50 eggs /10 ml urine were considered as cases with severe urinary schistosomiasis infection (World Health Organization, 1983).

Children aged between 5 and 15 years; who had no history of S. haematobium drug administration in the two weeks prior to screening, who have no  other serious chronic infection, and had ability to give blood and urine samples were included in the study.

Ethical clearance

The study protocol was reviewed and approved by the Ethical Review Committee of the Department of Biology, Addis  Ababa  University. Written  informed  consent  was consent was obtained from parents/caretakers of children and assent from the 15 years olds, after explaining the purpose and objective of the study.

Laboratory diagnosis

Urine analysis

Mid stream urine samples were collected between 10 a.m. and 2 p.m., the pick time of egg passage (World Health Organization, 1998). Children are provided plastic urine cups and asked to bring mid-stream urine. Each cup was given a serial number and the volume was recorded. Urine samples were analysed using the centrifugation method as described by Okanla (1991). Briefly, the samples were left to stand on the bench for about 30 min. Following this, the urine in each sample was drawn off leaving the last 10 ml in the bottle. The content of each bottle was shaken to suspend the sediment and was transferred into a 20 ml centrifuge tube. The tubes were centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the residue was put on a clean glass slide and examined under 10x objective lens of the microscope. Intensity of infection was estimated according to the number of eggs per 10 ml urine.

Determination of haemoglobin concentration

Peripheral blood was collected from a finger prick using a sterile blood lancet and analysed by portable, battery-operated HemoCue Hb 201 analyzer (HemoCue AB, Angel Holm, Sweden).

Blood group determination

Blood grouping test was done by standard hemagglutination techniques (Zoysa, 1985).

Briefly, 10 µl (approximately two drops) of whole blood were placed in two different places of a grease-free clean glass slide on which 10 µl of antisera for blood group A and B (ALBAclone® Anti-A, B, USA) was applied. The blood cells and the antigen were mixed with applicator stick. The slide was then tilted to detect for agglutination and the result recorded accordingly (Zoysa, 1985).

Treatment

Children who were positive for urinary schistosomiasis were treated based on the recommended drug regimen. A single dose of praziquantel (40 mg/kg body weight) was given to treat urinary schistosomiasis. Children with severe anemia were referred to the nearby health post and clinic for treatment and further follow up.

* Significant difference; † ANOVA; NA^≠, not applicable.

Table 2.  Percentage distribution of the ABO blood group types in the study categories.

Data analysis

All raw data was entered in to Microsoft excel data base system. The data then imported to SPSS version 15.0 software for analysis. Chi-square was used to determine association. Difference between means was analysed by ANOVA and odds ratios (OR) were calculated with 95% confidence interval (CI). Values were considered to be statistically significant when P-values were less than 0.05. 

RESULTS

Characteristics of the study participants

A total of 370 school children were included in this study, 95 were found to be infected with S. haematobium and the remaining 275 study participants were not infected by urinary schistosomiasis and are referred as healthy controls. A significant difference was observed in haemoglobin concentration between the mild schistosomiasis, 12.16 (1.39) gm/dl (mean (± SD), severe schistosomiasis, 11.06 (1.63) gm/dl (mean (± SD) and healthy control cases, 12.20 (2.15) gm/dl (mean (± SD) (P =.001) (Table1).

Percentage distribution of the ABO blood group types in the study categories

Out of the 48 severe schistosomiasis cases, 23 (47.9%)

were of blood group A, 11 (22.9 %) were of blood group B and 12 (25%) belonged to blood group O (Table 2).

In   the   mild   schistosomiasis   cases,  there  were  12

(25.5%), 6(12.5%), 2(4.3%) and 27(56.3%) blood group A, B, AB and O patients, respectively (Table 2). Blood group O was the dominant blood type in both mild schistosomiasis (56.3%) and healthy controls (53.1%) (Table 2).

The odds ratios and P values for the frequency of O and non-O blood group types between the three study categories

Compared to the mild schistosomiasis cases, a case of heavy urinary schistosomiasis was more than four times as likely to be of type A as to be of type O (SS vs. MS: O vs. A odds ratio 0.23, 95% confidence interval 1.62 to 11.42) and almost twice more likely to be of type AB as to be of type O (SS vs. MS: O vs. AB odds ratio 0.44, 95% confidence interval 0.28-17.91) (Table 3). As compared to the healthy control cases, the case of severe urinary schistosomiasis was more likely to be of type A (SS vs. HC: O vs. A, odds ratio (OR) 0.30, 95% confidence interval (CI) 1.58 to 7.04) and B (SS vs. HC: O vs. B, OR 0.28, 95% CI 1.48 to 8.84) than type O (Table 3).

Mean egg counts, haemoglobin concentration and ABO blood groups in children with urinary schistosomiasis

Intensity of S. haematobium infection was significantly

higher in children with blood group A (mean (± SD) 1.93(0.45) egg per 10 ml urine) than blood group O (mean (± SD) 1.63(0.39) egg per 10 ml urine) children (P=0.003). Although the mean egg count in children

^X values shown are the odds ratios, [95% CI], (P-value).

Table 4. Mean egg counts, haemoglobin concentration and ABO blood groups in children with urinary schistosomiasis.

* Significant difference; † ANOVA.

with blood group O (mean (± SD) 1.63 (0.39) egg per 10 ml urine) was higher than in those with blood group AB (mean (± SD) 1.45 (0.21) egg per 10 ml urine), the difference was not significant (P = 0.54) (Table 4). Furthermore, blood group O children (mean (± SD) 11.58 (1.62) gm/dl) were found to have lower mean haemoglobin concentration than children with blood group A (mean (± SD) 11.97 (1.72) gm/dl) but the difference was not statistically significant (P= 0.31) (Table 4).