Influence of ethnicity on pharmacogenetic variation in the Ghanaian population.

It has been well established that the frequencies of genomic variants can vary greatly between the populations of different countries. We sought to quantify the intra-population variability in Ghana to determine the value of genotyping studies done at a nationwide level. Further, we investigated the differences between the Ghanaian and other African populations to determine the quality of genomic representation provided by a small subgroup within the continent with regard to the general population. We genotyped 934 unrelated Ghanaian individuals for 15 single nucleotide polymorphisms (SNPs) from genes defined as clinically relevant based on their reported roles in the transport of, metabolism of, or as targets of the medicines listed in the World Health Organization Essential Medicines list. Populations within Ghana and between nations in Western Africa were genetically cohesive. In contrast, populations in other areas of Africa were genetically divergent. Gene allele frequency also differed significantly between the populations in African nations and the United States for several of the SNPs. These results demonstrate that national populations in similar geographic regions, like Africa, may have widely varying genetic allele frequencies for clinically relevant SNPs. Further genotyping studies of specific populations are necessary to provide the best medical care to all individuals.

Situation analysis of TB microscopy centres in Ghana.

SETTING: Public health laboratories in Ghana performing tuberculosis (TB) microscopy. OBJECTIVE: To assess the situation of the laboratories in terms of staff strength, technical skills, documentation, biosafety practices, equipment, supplies and disposal systems. DESIGN: Methods used for data collection were interviews using a structured questionnaire, informal observation of laboratory registers, disposal systems and safety measures for sputum handling. RESULTS: Of 114 laboratories visited between 2000 and 2001, 102 (89.5%) were performing TB microscopy. Of the staff working in the laboratories, 9% were medical technologists, 24% laboratory technicians, 37% laboratory assistants and 30% orderlies. Average false-negative and -positive rates were respectively 13% and 14%. Although most of the centres (85.3%) were using the recommended TB laboratory register for recording, in most cases they were not filled in accurately or completely. The majority of the available microscopes had mechanical or optical faults. Availability of other materials for smear preparation and staining ranged from 44% to 82%. The main methods employed for disposal of laboratory waste were burning and burying, but conditions were poor in most of the facilities visited. CONCLUSION: Training of laboratory personnel in TB microscopy and establishment of a quality assurance system are needed in Ghana.

Improving the laboratory diagnosis of TB in Ghana: the impact of a quality assurance system.

SETTING: Greater Accra region, Ghana. OBJECTIVE: To establish a pilot quality assurance (QA) system in sputum smear microscopy and to evaluate its impact. DESIGN: Quarterly supporting visits were paid to participating laboratories between 2000 and 2002. Fifteen examined slides were selected randomly from each laboratory during the visits and blindly re-assessed. Feedback was given promptly to the various laboratories. Training and stakeholder workshops were organised whenever necessary. RESULTS: General improvements in smear preparation and staining as well as the reading ability of the laboratory personnel included in the study were observed. The average marks for specimen quality, staining ability, smear cleanness, thickness, size and evenness increased from 64%, 79%, 69%, 46%, 67% and 60% in the last quarter of 2000 to 81%, 90%, 86%, 79%, 80% and 74%, respectively, 24 months after the establishment of the QA system. Within the same period, the rate of false-positives and -negatives decreased from respectively 14.8% and 20.5% to 0%, and agreements in positivity grade increased from 74% to 95%. The performance of the participating laboratories in keeping the laboratory registers up to date also improved. CONCLUSION: The QA system needs to be extended to the rest of the country.

HTLV-1 and other viral sexually transmitted infections in antenatal and gynaecological patients in Ghana.

BACKGROUND: The study was undertaken to determine the prevalence of infection with Human T cell lymphotrophic (leukemia) virus-1 (HTLV-1), Hepatitis B virus, Hepatitis C virus and Human Immunodeficiency Virus (HIV) in patients attending the antenatal and gynaecological outpatient clinics at Korle-Bu Teaching Hospital (KBTH). DESIGN: Prospective observational survey. Serum from each of the 517 participants was analysed for infection with Hepatitis B surface antigen with a latex agglutination test kit (Biotech Laboratories Ltd., Suffolk, United Kingdom), and tested for antibodies to Human Immunodeficiency Virus (HIV), Hepatitis C virus, and Human T cell lymphotrophic (leukemia) virus-1 (HTLV-1) with SERODIA passive-particle agglutination assay kits (FUJIREDIO Inc., Tokyo, Japan). The results were compared with reports from other institutions. SETTINGS: The Obstetrics and Gynaecology outpatient clinics of the Korle-bu Teaching Hospital, Accra, Ghana. The virology Unit of the Noguchi Memorial Institute for Medical Research (NMIMR), Accra, Ghana. RESULTS: The prevalence of infection with Hepatitis B surface antigen (HBsAg) was 16.8%, Hepatitis C antibody 5.2% and HTLV-1 2.7%. Twelve (6%) out of 199 participants who gave informed consent tested positive for HIV antibody. CONCLUSIONS: The study has demonstrated a high transmissible risk of HBV, HIV, HTLV-1, and HCV in Ghana and the necessity for antenatal screening for HBsAg to identify babies at risk of neonatal hepatitis B infection for appropriate intervention.

Pharmacogenetics of catechol-O-methyltransferase: frequency of low activity allele in a Ghanaian population.

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of neurotransmitters, catechol hormones and drugs such as levodopa and methyldopa. Ethnic differences in COMT activity have been observed in several populations. Previous studies suggest that the g1947G>A low activity allele is less common in individuals of African origin. COMT genotyping was performed using a mini-sequencing method in 195 healthy Ghanaians with a frequency of the homozygous g1947G>A of 6%. This study provides confirmation that the low activity COMT allele is less common in individuals of African origin. This finding may be important clinically with regards to the treatment of many neuropsychiatric disorders and in the pathophysiology of various human disorders including estrogen-induced cancers, Parkinson's disease, depression and hypertension.

Novel thymidylate synthase enhancer region alleles in African populations.

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A polymorphic tandem repeat sequence in the enhancer region of the TS promoter was previously described, where the triple repeat gives higher in vitro gene expression than a double repeat. We recently identified ethnic differences in allele frequencies between Caucasian and Asian populations. We now describe assessment of genotype and allele frequencies of the TS polymorphism in 640 African (African American, Ghanaian and Kenyan) and Caucasian (UK, USA) subjects. The double and triple repeat were the predominant alleles in all populations studied. The frequency of the triple repeat allele was similar between Kenyan (49%), Ghanaian (56%), African American (52%), American Caucasian (54%) and British Caucasian (54%) subjects. However, two novel alleles contained 4 and 9 copies of the tandem repeat. These novel alleles were found at a higher allele frequency in African populations (Kenyan 7%, Ghanaian 3%, African American 2%) than Caucasians (UK 1%, USA 0%). The novel alleles identified in this study decrease in frequency with Western migration, while the common alleles are relatively stable. This is a unique example suggesting the influence of multiple selection pressures within individual populations. Hum Mutat 16:528, 2000.

Analysis of the CYP2D6 gene mutations and their consequences for enzyme function in a West African population.

The data on differences in the metabolic handling of the CYP2D6 probe drugs sparteine and debrisoquine, and the relationship between phenotype and genotype and gene frequencies for several mutant CYP2D6 alleles in African populations are limited and sometimes controversial. Therefore, in a West African population (Ghana), we investigated (i) the phenotype for sparteine debrisoquine by phenotyping 201 individuals with both drugs and (iii) the genotype for CYP2D6 (n = 326) and debrisoquine (n = 201) oxidation, (ii) the coregulatory control of sparteine and alleles *3 and *4 in 133 individuals and for the alleles *1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *14, *16, *17, *2b, *2xN, *2bxN in 193 individuals. Of the 326 individuals phenotyped with sparteine, eight had a metabolic ratio (MR)sp > 20 corresponding to a poor metabolizer frequency of 2.5% [95% (confidence interval) CI = 1.06-4.77]. The prevalence of the poor metabolizer phenotype for debrisoquine oxidation was 3% (95% CI = 1.1-6.39) with six of the 201 individuals having a MR greater than 12.6. The distribution of the MR of sparteine was trimodal whereas MR of debrisoquine was unimodally distributed with a pronounced kurtosis. In individuals phenotyped with both drugs, there was a significant correlation between the MRs (r(s) = 0.63, P < 0.001). The CYP2D6 alleles *1, *2 and *17 were the most common functional alleles occurring with frequencies of 43.7, 10.6 and 27.7%, respectively. The three other observed functional alleles *2xN, *10 and *20 had much lower frequencies (1.6%, 3.1% and 0.3%, respectively). Of the eight non-functional alleles, only *4 (6.3%) and *5 (6.0%) could be found. The allele *5 occurred with the same frequency as in Caucasian populations (4.1%) but the *4 allele had a much lower frequency (Caucasians 19.5%). One individual with *1/*1 was a poor metabolizer for sparteine and debrisoquine indicating the existence of as yet unknown non-functional alleles in this West African population. Although the prevalence of poor metabolizers and the number of heterozygotes for non-functional alleles was much lower in Ghanaians, the median MRsp of 0.7 was significantly higher in this population compared with a median MRsp of 0.4 in Caucasians, indicating a lower metabolic clearance for CYP2D6 substrates in the West Africans. The lower metabolic activity in Ghanaians could not be explained solely by the high frequency of the *17 allele, which is associated with an impairment of CYP2D6 enzyme function. In addition, a higher median MRsp of 0.5 corresponding to metabolic clearance of 346 ml/min was observed among extensive metabolizers with the genotype *1/*1. Thus, compared with the median of MRsp = 0.28 (CLmet 573 ml/min) in Caucasians homozygous for *1, the metabolic clearance of sparteine was 40% lower on average in respective Ghanaians.

MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity.

P-glycoprotein (PGP), the product of the multidrug resistance gene (MDR1), acts as an energy-dependent efflux pump that exports its substrates out of the cell. PGP expression is an important factor regulating absorption of a wide variety of medications. It has also been associated with intrinsic and acquired cross resistance to a number of structurally unrelated anticancer drugs. A single nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene, C3435T, was recently correlated with PGP protein levels and substrate uptake. Individuals homozygous for the T allele have more than four-fold lower PGP expression compared with CC individuals. As overexpression of PGP has been associated with altered drug absorption, therapy-resistant malignancies, and lower concentrations of HIV-1 protease inhibitors, this SNP may provide a useful approach to individualize therapy. To facilitate clinical application throughout the world, 1280 subjects from 10 different ethnic groups were evaluated for this SNP using the polymerase chain reaction-restriction fragment length polymorphism assay and the genotype and allele frequency for each group were ascertained. Marked differences in genotype and allele frequency were apparent between the African populations and the Caucasian/Asian populations (P < 0.0001). The Ghanaian, Kenyan, African American and Sudanese populations studied had frequencies of 83%, 83%, 84% and 73%, respectively, for the C allele. The British Caucasian, Portuguese, South-west Asian, Chinese, Filipino and Saudi populations had lower frequencies of the C allele compared to the African group (48%, 43%, 34%, 53%, 59%, and 55%, respectively). The high frequency of the C allele in the African group implies overexpression of PGP and may have important therapeutic and prognostic implications for use of PGP dependent drugs in individuals of African origin.

Circadian variation of platelet 3H-imipramine binding, platelet serotonin content, and plasma cortisol in healthy volunteers.

Platelet 3H-imipramine binding, platelet serotonin content, and plasma cortisol were measured in 10 healthy female volunteers. Samples were taken on 5 occasions with a 6-hr interval during a 24-hr period within 1 week in May. Although there were individual variations in the biochemical parameters over a 24-hr period, we could not establish a clear common circadian rhythm for all subjects in platelet 3H-imipramine binding and platelet serotonin concentrations. During the night, there was a significant decrease in Bmax values and a slight, but not significant, increase in serotonin concentrations. A distinct circadian rhythm was obtained for plasma cortisol, with lowest values during the night.

Two approximately 300 kilodalton Plasmodium falciparum proteins at the surface membrane of infected erythrocytes.

Two very large Plasmodium falciparum proteins are identified as constituents of the infected erythrocyte membrane. Sera were obtained from Aotus monkeys that had been repeatedly infected with asexual P. falciparum from one of four strains. The capacity of these sera to block in vitro cytoadherence of infected erythrocytes and agglutinate intact infected cells was determined. The sera were also used to immunoprecipitate protein antigens from detergent extracts of 125I-surface labeled or biosynthetically radiolabeled infected erythrocytes. For each serum/antigen combination, precipitation of only one protein correlated with the ability of the serum to interfere with cytoadherence and agglutinate infected cells. This malarial protein, denoted Pf EMP 1 (P. falciparum-erythrocyte-membrane-protein 1) bore strain-specific epitope(s) on the cell surface and displayed size heterogeneity (Mr approximately 220,000-350,000). Pf EMP 1 was strongly labeled by cell-surface radioiodination but was a quantitatively very minor malarial protein. Pf EMP 1 was distinguished by its size, surface accessibility and antigenic properties from a more predominant malarial protein in the same size range (Pf EMP 2) that is under the infected erythrocyte membrane at knobs. Monoclonal antibodies and rabbit antisera raised against Pf EMP 2 were used to show that this size heterogeneous antigen was indistinguishable from the previously described MESA (mature parasite infected erythrocyte surface antigen), identified by precipitation with rabbit antisera raised against the MESA hexapeptide repeats. Antibodies raised against Pf EMP 2/MESA did not precipitate Pf EMP 1. We conclude that Pf EMP 1 is either directly responsible for the cytoadherence phenomenon, or is very closely associated with another as yet unidentified functional molecule. Pf EMP 2/MESA must have a structural property/function that is important under the host cell membrane.

Plasmodium falciparum: sensitivity to chloroquine in vivo in three ecological zones in Ghana.

4690 children aged 6-15 years in 5 urban and 4 rural communities in 3 ecological zones in Ghana were screened from June 1988 to December 1990 to provide suitable candidates for the World Health Organization standard in vivo test for susceptibility of Plasmodium falciparum to chloroquine. 1880 (40.1%) had parasitaemia, mostly (83.7-98.6%) due to P. falciparum infection. Of the 626 in vivo tests performed, 570 (91.1%) showed sensitivity to chloroquine and 56 (8.9%) responses were classified as resistant to chloroquine at RI (5.1%) and RII (3.8%). The resistance responses were commonest (17.1-22.7%) in the coastal zone, followed by the savanna zone (8.6-10.0%), and lowest in the forest zone (3.1-6.3%). The RII responses occurred mainly in communities in the coastal zone. There was no RIII resistance in any zone. The pattern of RI (early) and RII responses of P. falciparum to chloroquine in this study suggested an increase in sensitivity, or a reduction in resistance, of P. falciparum to chloroquine from the coast to the forest and northern savanna zones, and from the urban to the rural communities in each zone in Ghana.

Comparative trial of oral versus intramuscular chloroquine in children with cerebral malaria.

One hundred and thirteen children aged 12 years or less with cerebral malaria in Accra, Ghana were treated with chloroquine either with a low dose regime of 3.5 mg/kg 8-hourly intramuscularly, or orally by nasogastric tube, in a standard regime, both to a total of 25 mg/kg body weight. There was no obvious difference in outcome in the 2 treatment groups. The overall mortality of 5.3% (5.9% and 4.4% in the oral and intramuscular treatment groups respectively) was similar to that seen 10 years ago in this hospital. The average parasite clearance time had increased to 61 h, compared to 41 h noted 10 years ago. The incidence of hypoglycaemia (3%) was very low compared to studies in other malaria endemic areas. The reason for this is not clear but it could have contributed to the low mortality. Neurological deficits were seen on day 14 in 7.8% of patients. Parasitaemia recurred within 14 d in 22% of surviving patients, confirming the presence of RI/RII chloroquine resistance in Accra.

Effect of training on the clinical management of malaria by medical assistants in Ghana.

Malaria accounts for over 40% of all outpatient consultations in Ghana. A common drug use problem associated with its treatment with chloroquine is over- and under-dosage and a preference for the intramuscular route of administration. Inadequate treatment is an important factor in the selection of resistant strains of malaria parasites. To ensure the proper management of diseases at health centres the Ministry of Health instituted an in-service training programme for medical assistants in 1987. We evaluated the effect of this training on the clinical management of malaria using a quasi-experimental design. Three methods of data collection were used; prescription survey, assessment questionnaires and focus group discussions. Our findings revealed that gains in knowledge following the training had deteriorated within a year. There was also a discrepancy between knowledge and practice of malaria treatment. This was shown by over- and under-dosing of chloroquine in children and adults respectively. There was also overwhelming preference (85% of all cases) for injections and a high tendency towards polypharmacy (average of five drugs per visit). The motivating reasons for these were mainly socio-cultural and included patient demand and attitudes, prescriber self interests and stereotypes and the daily practical challenges of the community. While paying greater attention to supervision of clinical work at health posts, consideration must be given to socio-cultural context of drug use in any such future training programmes if rational use of drugs is to be achieved.

A strategy for promoting improved pharmaceutical use: the International Network for Rational Use of Drugs.

Over the last decade, pharmaceutical selection, procurement, distribution, and financing have improved as a result of essential drugs programs. However, despite improved availability, pharmaceuticals are frequently used irrationally. The International Network for the Rational Use of Drugs (INRUD) has been established to help address this problem. The Network joins core groups of researchers from four African and three Asian countries with support groups in Boston, Sweden, WHO, and Australia. The activities of the Network are supported by multilateral, bilateral, foundation donors and by Management Sciences for Health. INRUD functions as a participatory organization in which members are involved in decision-making. The primary objective of the Network is to identify through a coordinated set of country-based research projects a set of effective interventions to recommend as policy options for the promotion of rational drug use. In developing these research projects, INRUD stresses the importance of a multi-disciplinary perspective for adequately understanding the reasons underlying inappropriate use of drugs. To better enable country groups to utilize strong research methodologies and to blend the strengths of multiple disciplines effectively, a major activity of the Network thus far has been the building of local research capacity.

Survey of antibiotic prescribing pattern in government health facilities of the Wassa west district of Ghana.

Antibiotic prescribing patterns was studied from 700 retrospective outpatient clinical records from seven government health facilities in the Wassa West district of Ghana. Prescribing patterns were compared between the district hospital and six health centres. The percentage of patients receiving one or more antibiotics was significantly more at the health centres(60.7%) than at the hospital(41.0%) (chi 2 = 13.6; p < 0.001). The average number of antibiotics prescribed per patient was 1.4 and 1.1 respectively. The commonest antibiotics prescribed were procaine penicillin, cotrimoxazole, benzylpenicillin, metronidazole and amoxycillin. Malaria, upper respiratory infections, soft tissue infections and diarrhoeal diseases were the commonest indications for antibiotic use. Factors such as the availability of diagnostic facilities, type of prescriber, lack of refresher training and patient demand were considered to significantly influence antibiotic prescribing.

Knowledge and management of malaria in Dar es Salaam, Tanzania.

A study of knowledge and management of malaria was undertaken prospectively in 20 dispensaries, 20 drug stores, 120 patients attending dispensaries and 120 customers at drug stores in Dar es Salaam, Tanzania. This was a descriptive study where two different questionnaires were developed and administered to the target groups in oral interview. All the respondents were aged 14 years and above. The results of the interview showed that the knowledge of rural medical aides (RMA's) on signs and symptoms of malaria and which drugs cure malaria was satisfactory. However, only 65% of the RMA's could remember the correct dose of chloroquine for an adult. Although the knowledge of drug sellers on signs and symptoms of malaria was adequate, 45% of them did not know the correct dose of chloroquine. In view of the fact that only 30% of patients and 20% of shoppers knew the correct dose of chloroquine for adults, it appears that their management of malaria is inadequate. To improve the management of malaria at dispensary and drug store level, there is a need to introduce treatment charts and/or guidelines and the Ministry of Health should promote health education to the public.

PIP: In early 1992 in Tanzania, trained research assistants interviewed 20 rural medical aides (RMAs), 20 drug sellers, 120 patients at 20 dispensaries, and 120 customers of drug stores, all in Dar es Salaam, to examine knowledge and practices of health workers, drug sellers, and patients concerning malaria. The RMAs had adequate knowledge of the signs and symptoms of malaria (e.g., 90% for fever, 85% for headache, and 80% for painful joints). The drug sellers also had adequate knowledge of signs and symptoms (e.g., 80% for fever, 45% for headache, and 50% for painful joints). Even though chloroquine-resistant strains of Plasmodium falciparum malaria exist in Tanzania, all RMAs and most drug sellers (85%) believed that chloroquine could cure malaria. Further, it is the only antimalarial that the Ministry of Health provides its dispensaries. Just 65% of RMAs knew the correct dosage of chloroquine for people 14 years old and older. An even lower percentage of drug sellers knew the correct dosage (50%). Just 33.7% of patients and 22.5% of customers knew the correct dosage of chloroquine. An inadequate supply of chloroquine was available at 15% of dispensaries and 30% of drug stores. RMAs sometimes gave patients too few chloroquine tablets for a full course of therapy. Other drugs mentioned by RMAs to treat malaria were quinine, amodiaquine, cotrimoxazole, halofantrine, pyrimethamine/sulphalene, and pyrimethamine/sulphadoxine. Those mentioned by drug sellers included aspirin plus chloroquine, cotrimoxazole, pyrimethamine/sulphadoxine, and traditional medicines. These findings suggest that irrational drug therapy of malaria promotes resistant strains and prolongs the duration of illness. Introduction of treatment charts and health education promotion to the public are needed to improve malaria management at dispensaries and drug stores.

In vitro responses of P. falciparum parasites to chloroquine, amodiaquine and quinine in two ecological zones in Ghana.

Blood specimens were taken from 318 school children with at least 1,000 and not more than 80,000 P. falciparum asexual parasites per microliter of blood for a 30 hour in vitro microtest of P. falciparum asexual parasites responses to chloroquine, amodiaquine and quinine. The study was conducted in primary schools in four urban and three rural communities in the costal and forest zones in Ghana between June 1988 and December 1990. Chloroquine resistance was present in 58.7 pc (54/92) and 3.9 pc (4/103) of the successful in vitro tests in the coastal and forest zones respectively. Resistance to amodiaquine was recorded in 28.6 pc (12/42) of the successful tests in coastal zone. There was no resistance to quinine in any of the ecological zones. Concentrations of the three drugs in pmol required for 90 pc inhibition of schizont maturation were generally higher in communities in the coastal zone than those in the forest zone. The results suggest an increase in sensitivity or a reduction in resistance of P. falciparum to the drugs from the coast to the forest zone.

A 1-day survey of drug prescribing patterns in the District General Hospital of the Wassa West District of Ghana.

A 1-day prevalence survey of the drug prescribing patterns for 45 in-patients of the Tarkwa Government Hospital was undertaken in November 1995. An average of 3.6 drugs were prescribed per patient. The commonest prescribed drugs were paracetamol, chloroquine, amoxycillin, gentamicin and ampicillin. Of 162 drug items prescribed, 93.2% were on the national Essential Drugs List (EDL) and 61.1% were prescribed by their generic names. Over 60% of the patients received one or more injectable drugs or antibiotics. The use of injectable drugs on the wards ranged from nil on the male surgical ward to 90.9% on the male medical ward. The most frequently prescribed injectable drugs were gentamicin, ampicillin, chloroquine, benzylpenicillin, dextrose and pethidine. The main indications for antibiotic use were post-Caesarean delivery, pneumonia, accidents, abscesses and meningitis. The findings provide the basis for monitoring drug prescribing patterns and for evaluating any interventions to improve prescribing behaviour and the treatment of diseases.

A study of prescribing patterns with special reference to drug use indicators in Dar es Salaam Region, Tanzania.

Drug prescribing patterns were studied in 720 retrospective and 779 prospective outpatient prescriptions from 20 dispensaries in Dar es Salaam region, and these revealed a mean drug exposure of 2.0 and 2.3, respectively. The percentage of patients leaving the dispensaries with no prescribed drugs was 1.3% and 0.7%, respectively. Prescriptions containing antibiotics were 36.8% (retrospective) and 39.8% (prospective), while injections accounted for 24.6% and 34% of the total encounters, respectively. Over 70% of prescriptions conformed to the Tanzania essential drug list (EDP) and/or standard treatment guidelines and consisted of 83.9% and 79.1% generic prescriptions, respectively. Interestingly, only 15% of the surveyed dispensaries had an EDP book and/or calendar. Despite the consulting and dispensing times being short (2.98 min and 77.7 s, respectively), 70% of the patients could remember the dosing instructions. Only 64% of the patients had a minimum physical examination.