Background In view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in gastroesophageal reflux disease, (GERD), we investigated the hypothesis that these changes result from a disordered myoelectric activity of the esophagus.
Background
In view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in gastroesophageal reflux disease, (GERD), we investigated the hypothesis that these changes result from a disordered myoelectric activity of the esophagus.Methods The electric activity of the esophagus (electroesophagogram, EEG) was studied in 27 patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 years) and 10 healthy volunteers as controls (6 men, 4 women, mean age 41.4 ± 4.9 years). According to the Feussner scoring system, 7 patients had a mild (score 1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. One electrode was applied to the upper third and a second to the lower third of the esophagus, and the electric activity was recorded. The test was repeated after the upper electrode had been moved to the mid-esophagus.
Methods
The electric activity of the esophagus (electroesophagogram, EEG) was studied in 27 patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 years) and 10 healthy volunteers as controls (6 men, 4 women, mean age 41.4 ± 4.9 years). According to the Feussner scoring system, 7 patients had a mild (score 1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. One electrode was applied to the upper third and a second to the lower third of the esophagus, and the electric activity was recorded. The test was repeated after the upper electrode had been moved to the mid-esophagus.Results The EEG of the healthy volunteers showed slow waves and exhibited the same frequency, amplitude and conduction velocity from the 2 electrodes of the individual subject, regardless of their location in the upper, middle or lower esophagus. Action potentials occurred randomly. In GERD patients, score 1 exhibited electric waves' variables similar to those of the healthy volunteers. In score 2, the waves recorded irregular rhythm and lower variables than the controls. Score 3 showed a "silent" EEG without waves.
Results
The EEG of the healthy volunteers showed slow waves and exhibited the same frequency, amplitude and conduction velocity from the 2 electrodes of the individual subject, regardless of their location in the upper, middle or lower esophagus. Action potentials occurred randomly. In GERD patients, score 1 exhibited electric waves' variables similar to those of the healthy volunteers. In score 2, the waves recorded irregular rhythm and lower variables than the controls. Score 3 showed a "silent" EEG without waves.Conclusion The electric activity in GERD exhibited 3 different patterns depending on the stages of GERD. Score 1 exhibited a normal EEG which apparently denotes normal esophageal motility. Score 2 recorded irregular electric waves variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" EEG was recorded with probably no acid clearance. It is postulated that the interstitial cells of Cajal which are the electric activity generators, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The EEG seems to have the potential to act as an investigative tool in the diagnosis of GERD stages.
Conclusion
The electric activity in GERD exhibited 3 different patterns depending on the stages of GERD. Score 1 exhibited a normal EEG which apparently denotes normal esophageal motility. Score 2 recorded irregular electric waves variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" EEG was recorded with probably no acid clearance. It is postulated that the interstitial cells of Cajal which are the electric activity generators, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The EEG seems to have the potential to act as an investigative tool in the diagnosis of GERD stages.

Background Reflux esophagitis is a multifactorial disease that entails the reflux of gastric contents into the esophageal lumen [ 1 ]. Esophagitis develops when noxious substances in the refluxate have sufficient time to get in contact with the esophageal mucosa and then prevail over structural and functional defenses [ 2 ] The mechanisms involved in the defense of the esophagus against the gastric acid pepsin comprise the antireflux barriers, luminal clearance and epithelial resistance [ 3 - 6 ]. The antireflux barriers and the luminal clearance mechanism by peristalsis represent motor components of the reflux disease. The non-motility elements include salivary and esophageal submucosal glands. Abnormal esophageal motility results in an increased exposure of the esophageal mucosa to gastric contents. Progressing severity of the reflux disease is associated with failing primary peristalsis [ 7 - 9 ]. The failure rate in patients with mild and severe GERD is 25% and 36%, respectively [ 7 ]. The amplitude of peristaltic contractions in the esophagus is significantly lower in the esophagitis patients than in the controls [ 6 - 9 ], and is inversely related to esophagitis severity [ 7 , 10 ]. The duration of contractions was variable but the propagation velocity was unequivocally slower in esophagitis patients than in controls [ 7 , 8 , 11 ]. Previous studies have shown that the esophagus possesses electric activity presenting as slow waves (SWs) followed or superimposed by fast activity spikes or action potentials (APs) [ 12 , 13 ]. Action potentials were associated with a rise in the intraesophageal pressure. Balloon distension of the esophagus effected an increase in the electric activity proximally to the balloon and a decrease distally [ 12 ]. The caudad direction of the SWs and APs was evidenced when after esophageal myotomy the potentials appeared proximally but not distally to the cut in the experimental animal [ 12 ]. This suggested also the presence of a pacemaker in the cervical esophagus which might initiate the electric waves[ 12 ].In achalasia of the esophagus, three electroesophagographic patterns were identified: bradyesophagia, esophagoarrhythmia and "silent" electroesophagogram [ 13 ]. The three patterns seem to represent different stages in one pathologic process. In view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in GERD, we hypothesized that these findings result from a disordered myoelectric activity of the esophagus. This hypothesis was investigated in the current study.
Background
Reflux esophagitis is a multifactorial disease that entails the reflux of gastric contents into the esophageal lumen [ 1 ]. Esophagitis develops when noxious substances in the refluxate have sufficient time to get in contact with the esophageal mucosa and then prevail over structural and functional defenses [ 2 ] The mechanisms involved in the defense of the esophagus against the gastric acid pepsin comprise the antireflux barriers, luminal clearance and epithelial resistance [ 3 - 6 ]. The antireflux barriers and the luminal clearance mechanism by peristalsis represent motor components of the reflux disease. The non-motility elements include salivary and esophageal submucosal glands.Abnormal esophageal motility results in an increased exposure of the esophageal mucosa to gastric contents. Progressing severity of the reflux disease is associated with failing primary peristalsis [ 7 - 9 ]. The failure rate in patients with mild and severe GERD is 25% and 36%, respectively [ 7 ]. The amplitude of peristaltic contractions in the esophagus is significantly lower in the esophagitis patients than in the controls [ 6 - 9 ], and is inversely related to esophagitis severity [ 7 , 10 ]. The duration of contractions was variable but the propagation velocity was unequivocally slower in esophagitis patients than in controls [ 7 , 8 , 11 ].Previous studies have shown that the esophagus possesses electric activity presenting as slow waves (SWs) followed or superimposed by fast activity spikes or action potentials (APs) [ 12 , 13 ]. Action potentials were associated with a rise in the intraesophageal pressure. Balloon distension of the esophagus effected an increase in the electric activity proximally to the balloon and a decrease distally [ 12 ]. The caudad direction of the SWs and APs was evidenced when after esophageal myotomy the potentials appeared proximally but not distally to the cut in the experimental animal [ 12 ]. This suggested also the presence of a pacemaker in the cervical esophagus which might initiate the electric waves[ 12 ].In achalasia of the esophagus, three electroesophagographic patterns were identified: bradyesophagia, esophagoarrhythmia and "silent" electroesophagogram [ 13 ]. The three patterns seem to represent different stages in one pathologic process.In view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in GERD, we hypothesized that these findings result from a disordered myoelectric activity of the esophagus. This hypothesis was investigated in the current study.Methods Subjects Twenty seven patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 SD years, range 36–48) were enrolled in the study. The diagnosis was confirmed by 24-hour pH monitoring, endoscopy and esophageal motility test. According to Feussner's scoring system [ 14 ], seven patients had a mild (score1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. Score 1 had mild heartburn with mild chest pain but no regurgitation, dysphagia, hiatus hernia or mucosal changes. Exposure time to pH was <4.4–8%. Score 2 had moderate heartburn, moderate chest pain, regurgitation after large meals, small hiatus hernia, and isolated erosive mucosal lesions. Score 3 had severe heartburn, severe chest pain, regurgitation, occasional dysphagia, hiatus hernia, and esophageal ulcers. The study also included 10 healthy volunteers (6 men, 4 women, mean age 41.4 ± 4.9 SD years, range 35–50) who had no reflux esophagitis. They had no gastrointestinal complaints in the past or at the time of enrollment in the study. Physical examination of both the patients and healthy volunteers had normal findings. The results of laboratory work comprising blood count, renal and hepatic function tests as well as electrocardiogram were unremarkable. The studied subjects gave an informed consent after having been fully informed about the nature of the study, the tests to be done and their role in the study. The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine. Methods The electric activity of the esophagus was recorded in the patients with GERD and the healthy volunteers. We used a monopolar silver-silver chloride electrodes of 0.8 mm diameter (Smith-Kline Beecham, Los Angeles, CA, USA) introduced through a 6F catheter (Rubber Industries Ltd., London, UK) with the electrode protruding by 1 cm from the catheter tip. The catheter was attached to the esophageal mucosa by negative pressure suction of 50 to 100 mmHg which was maintained during the test. Two electrodes were introduced into the esophagus by means of an endoscope and fixed to the esophageal mucosa by suction; one electrode was applied to the upper third, and the second to the lower third of the esophagus, and the electric activity was recorded. The upper electrode was then transferred to the middle third of the esophagus and recordings of the electric activity from the middle and lower third electrodes were performed. Signals from the electrodes were fed into an AC amplifier with a frequency response within ± 3 dB from 0.016 Hz to 1 kHz; they were displayed on a recorder at a sensitivity of 1 mV/cm. A metal disc applied to the abdominal skin served as the indifferent electrode. A strain gauge respiration transducer was attached to the thoracic wall for respiratory artefacts. We allowed the esophagus a 30 minute period to adapt to the electrodes applied to its wall, before we started a 120-min recording session for each subject. The results were analyzed statistically using the Student's t test. Differences assumed significance at p < 0.05 and values were given as the mean ± standard deviation (SD).
Methods
Subjects Twenty seven patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 SD years, range 36–48) were enrolled in the study. The diagnosis was confirmed by 24-hour pH monitoring, endoscopy and esophageal motility test. According to Feussner's scoring system [ 14 ], seven patients had a mild (score1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. Score 1 had mild heartburn with mild chest pain but no regurgitation, dysphagia, hiatus hernia or mucosal changes. Exposure time to pH was <4.4–8%. Score 2 had moderate heartburn, moderate chest pain, regurgitation after large meals, small hiatus hernia, and isolated erosive mucosal lesions. Score 3 had severe heartburn, severe chest pain, regurgitation, occasional dysphagia, hiatus hernia, and esophageal ulcers. The study also included 10 healthy volunteers (6 men, 4 women, mean age 41.4 ± 4.9 SD years, range 35–50) who had no reflux esophagitis. They had no gastrointestinal complaints in the past or at the time of enrollment in the study. Physical examination of both the patients and healthy volunteers had normal findings. The results of laboratory work comprising blood count, renal and hepatic function tests as well as electrocardiogram were unremarkable. The studied subjects gave an informed consent after having been fully informed about the nature of the study, the tests to be done and their role in the study. The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.
Subjects
Twenty seven patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 SD years, range 36–48) were enrolled in the study. The diagnosis was confirmed by 24-hour pH monitoring, endoscopy and esophageal motility test. According to Feussner's scoring system [ 14 ], seven patients had a mild (score1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. Score 1 had mild heartburn with mild chest pain but no regurgitation, dysphagia, hiatus hernia or mucosal changes. Exposure time to pH was <4.4–8%. Score 2 had moderate heartburn, moderate chest pain, regurgitation after large meals, small hiatus hernia, and isolated erosive mucosal lesions. Score 3 had severe heartburn, severe chest pain, regurgitation, occasional dysphagia, hiatus hernia, and esophageal ulcers.The study also included 10 healthy volunteers (6 men, 4 women, mean age 41.4 ± 4.9 SD years, range 35–50) who had no reflux esophagitis. They had no gastrointestinal complaints in the past or at the time of enrollment in the study.Physical examination of both the patients and healthy volunteers had normal findings. The results of laboratory work comprising blood count, renal and hepatic function tests as well as electrocardiogram were unremarkable. The studied subjects gave an informed consent after having been fully informed about the nature of the study, the tests to be done and their role in the study. The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.Methods The electric activity of the esophagus was recorded in the patients with GERD and the healthy volunteers. We used a monopolar silver-silver chloride electrodes of 0.8 mm diameter (Smith-Kline Beecham, Los Angeles, CA, USA) introduced through a 6F catheter (Rubber Industries Ltd., London, UK) with the electrode protruding by 1 cm from the catheter tip. The catheter was attached to the esophageal mucosa by negative pressure suction of 50 to 100 mmHg which was maintained during the test. Two electrodes were introduced into the esophagus by means of an endoscope and fixed to the esophageal mucosa by suction; one electrode was applied to the upper third, and the second to the lower third of the esophagus, and the electric activity was recorded. The upper electrode was then transferred to the middle third of the esophagus and recordings of the electric activity from the middle and lower third electrodes were performed. Signals from the electrodes were fed into an AC amplifier with a frequency response within ± 3 dB from 0.016 Hz to 1 kHz; they were displayed on a recorder at a sensitivity of 1 mV/cm. A metal disc applied to the abdominal skin served as the indifferent electrode. A strain gauge respiration transducer was attached to the thoracic wall for respiratory artefacts. We allowed the esophagus a 30 minute period to adapt to the electrodes applied to its wall, before we started a 120-min recording session for each subject. The results were analyzed statistically using the Student's t test. Differences assumed significance at p < 0.05 and values were given as the mean ± standard deviation (SD).
Methods
The electric activity of the esophagus was recorded in the patients with GERD and the healthy volunteers. We used a monopolar silver-silver chloride electrodes of 0.8 mm diameter (Smith-Kline Beecham, Los Angeles, CA, USA) introduced through a 6F catheter (Rubber Industries Ltd., London, UK) with the electrode protruding by 1 cm from the catheter tip. The catheter was attached to the esophageal mucosa by negative pressure suction of 50 to 100 mmHg which was maintained during the test.Two electrodes were introduced into the esophagus by means of an endoscope and fixed to the esophageal mucosa by suction; one electrode was applied to the upper third, and the second to the lower third of the esophagus, and the electric activity was recorded. The upper electrode was then transferred to the middle third of the esophagus and recordings of the electric activity from the middle and lower third electrodes were performed. Signals from the electrodes were fed into an AC amplifier with a frequency response within ± 3 dB from 0.016 Hz to 1 kHz; they were displayed on a recorder at a sensitivity of 1 mV/cm. A metal disc applied to the abdominal skin served as the indifferent electrode. A strain gauge respiration transducer was attached to the thoracic wall for respiratory artefacts. We allowed the esophagus a 30 minute period to adapt to the electrodes applied to its wall, before we started a 120-min recording session for each subject.The results were analyzed statistically using the Student's t test. Differences assumed significance at p < 0.05 and values were given as the mean ± standard deviation (SD).Results No adverse side effects were encountered during or after the performance of the tests and all the subjects were evaluated. There was no difficulty in applying the electrodes to the esophageal mucosa. We found that a suction pressure of 50–60 mmHg was sufficient to keep the electrode fixed to the esophageal mucosa during the testing period in most of the subjects; in few cases we had to increase the suction pressure to 100 mm to keep the electrodes in position. Applying the aforementioned pressures, we encountered neither migration nor detachment of the electrodes during the entire test. We met no mucosal bleeding, tears or ulcers during application or after removal of the electrodes either. Electroesophagogram in healthy subjects Monophasic negatively deflected SWs were recorded from the 2 electrodes of each subject of all the studied individuals (fig.  1 ). They had an unvariable shape in all recordings from the same site. The frequency, amplitude and conduction velocity were constant in the individual subject. The SWs in each individual exhibited the same frequency, amplitude and regular rhythm from both electrodes (fig.  1 ), regardless of their location in the upper, middle or lower third of the esophagus. The mean and range of frequency, amplitude and conduction velocity of the 10 healthy volunteers are displayed in table  1 . These values were reproducible from the electrodes in the upper, middle or lower third of the esophagus. Bursts of APs representing fast activity spikes were recorded (fig.  1 ). They followed or were superimposed over the SWs; they occurred randomly and their frequency was inconsistent in each subject. Figure 1 Electroesophagogram of a healthy volunteer showing slow waves with regular rhythm and random action potentials. Table 1 The frequency, amplitude and conduction velocity of the slow waves of the healthy volunteers and patients with gastroesophageal reflux disease (GERD) + Slow waves Frequency c/m Amplitude (mV) Velocity (cm/s) Mean Range Mean Range Mean Range Volunteers 5.2 ± 1.3 4 – 7 0.52 ± 0.1 0.4 – 0.6 4.8 ± 0.7 3.5 – 6.1 Score 1 GERD 4.8 ± 1.2 4 – 6 • 0.49 ± 0.1 • 0.35 – 0.6 4.6 ± 0.7 • 4.1 – 5.9 Score 2 GERD Irregular Score 3 GERD Absent waves +  values were given as the mean ± SD •  p > 0.05 p values of the patients were compared to those of the healthy volunteers Electroesophagogram in GERD patients In score 1 GERD, the electric waves' variables were similar to those of the healthy volunteers in all the subjects (p > 0.05, fig  2 ). The SWs were monophasic and negatively deflected and had a regular rhythm (fig  2 ). The frequency, amplitude and conduction velocity are shown in table  1 . Action potentials were randomly recorded following or superimposed over the SWs. This electromyographic pattern was similar from the 2 recording electrodes of the individual subject and was reproducible during the recording period. Figure 2 Electroesophagogram of a patient with score 1 gastroesophageal reflux disease showing slow waves with regular rhythm and random action potentials. Score 2 GERD patients exhibited a different electromyographic pattern. The SWs had an irregular rhythm with varying but lower frequency, amplitude and conduction velocity compared to the normal controls (fig.  3 ). The APs occurred randomly and were less frequent than in the normal recordings (fig.  3 ). The SWs and APs differed from one electrode to the other of the same subject and were variable during the recording period. In 8 score 3 GERD patients, the electrodes did not register electric waves; neither SWs nor APs were recorded. A "silent" electromyographic pattern was registered (fig.  4 ); this picture was reproducible during the recording period. In the remaining 2 patients; occasional SWs were recorded that were inconsistent and different from the 2 electrodes of the same patient (fig.  5 ); no APs were recorded at any time during the recording period (fig.  5 ). Figure 3 Electroesophagogram of a patient with score 2 gastroesophageal reflux disease exhibiting slow waves with irregular rhythm and varying frequency, amplitude and conduction velocity from the same electrode and between the 2 electrodes of the same subject. Figure 4 Electroesophagogram of a patient with score 3 gastroesophageal reflux disease recording no electric activity: a "silent" electroesophagogram. Figure 5 Electroesophagogram of a patient with score 3 gastroesophageal reflux disease recording occasional slow waves with no action potentials.
Results
No adverse side effects were encountered during or after the performance of the tests and all the subjects were evaluated. There was no difficulty in applying the electrodes to the esophageal mucosa. We found that a suction pressure of 50–60 mmHg was sufficient to keep the electrode fixed to the esophageal mucosa during the testing period in most of the subjects; in few cases we had to increase the suction pressure to 100 mm to keep the electrodes in position. Applying the aforementioned pressures, we encountered neither migration nor detachment of the electrodes during the entire test. We met no mucosal bleeding, tears or ulcers during application or after removal of the electrodes either.Electroesophagogram in healthy subjects Monophasic negatively deflected SWs were recorded from the 2 electrodes of each subject of all the studied individuals (fig.  1 ). They had an unvariable shape in all recordings from the same site. The frequency, amplitude and conduction velocity were constant in the individual subject. The SWs in each individual exhibited the same frequency, amplitude and regular rhythm from both electrodes (fig.  1 ), regardless of their location in the upper, middle or lower third of the esophagus. The mean and range of frequency, amplitude and conduction velocity of the 10 healthy volunteers are displayed in table  1 . These values were reproducible from the electrodes in the upper, middle or lower third of the esophagus. Bursts of APs representing fast activity spikes were recorded (fig.  1 ). They followed or were superimposed over the SWs; they occurred randomly and their frequency was inconsistent in each subject. Figure 1 Electroesophagogram of a healthy volunteer showing slow waves with regular rhythm and random action potentials. Table 1 The frequency, amplitude and conduction velocity of the slow waves of the healthy volunteers and patients with gastroesophageal reflux disease (GERD) + Slow waves Frequency c/m Amplitude (mV) Velocity (cm/s) Mean Range Mean Range Mean Range Volunteers 5.2 ± 1.3 4 – 7 0.52 ± 0.1 0.4 – 0.6 4.8 ± 0.7 3.5 – 6.1 Score 1 GERD 4.8 ± 1.2 4 – 6 • 0.49 ± 0.1 • 0.35 – 0.6 4.6 ± 0.7 • 4.1 – 5.9 Score 2 GERD Irregular Score 3 GERD Absent waves +  values were given as the mean ± SD •  p > 0.05 p values of the patients were compared to those of the healthy volunteers
Electroesophagogram in healthy subjects
Monophasic negatively deflected SWs were recorded from the 2 electrodes of each subject of all the studied individuals (fig.  1 ). They had an unvariable shape in all recordings from the same site. The frequency, amplitude and conduction velocity were constant in the individual subject. The SWs in each individual exhibited the same frequency, amplitude and regular rhythm from both electrodes (fig.  1 ), regardless of their location in the upper, middle or lower third of the esophagus. The mean and range of frequency, amplitude and conduction velocity of the 10 healthy volunteers are displayed in table  1 . These values were reproducible from the electrodes in the upper, middle or lower third of the esophagus. Bursts of APs representing fast activity spikes were recorded (fig.  1 ). They followed or were superimposed over the SWs; they occurred randomly and their frequency was inconsistent in each subject.The frequency, amplitude and conduction velocity of the slow waves of the healthy volunteers and patients with gastroesophageal reflux disease (GERD) +Slow wavesFrequency c/mAmplitude (mV)Velocity (cm/s)MeanRangeMeanRangeMeanRangeVolunteersScore 1 GERDScore 2 GERDScore 3 GERD+  values were given as the mean ± SD•  p > 0.05p values of the patients were compared to those of the healthy volunteersElectroesophagogram in GERD patients In score 1 GERD, the electric waves' variables were similar to those of the healthy volunteers in all the subjects (p > 0.05, fig  2 ). The SWs were monophasic and negatively deflected and had a regular rhythm (fig  2 ). The frequency, amplitude and conduction velocity are shown in table  1 . Action potentials were randomly recorded following or superimposed over the SWs. This electromyographic pattern was similar from the 2 recording electrodes of the individual subject and was reproducible during the recording period. Figure 2 Electroesophagogram of a patient with score 1 gastroesophageal reflux disease showing slow waves with regular rhythm and random action potentials. Score 2 GERD patients exhibited a different electromyographic pattern. The SWs had an irregular rhythm with varying but lower frequency, amplitude and conduction velocity compared to the normal controls (fig.  3 ). The APs occurred randomly and were less frequent than in the normal recordings (fig.  3 ). The SWs and APs differed from one electrode to the other of the same subject and were variable during the recording period. In 8 score 3 GERD patients, the electrodes did not register electric waves; neither SWs nor APs were recorded. A "silent" electromyographic pattern was registered (fig.  4 ); this picture was reproducible during the recording period. In the remaining 2 patients; occasional SWs were recorded that were inconsistent and different from the 2 electrodes of the same patient (fig.  5 ); no APs were recorded at any time during the recording period (fig.  5 ). Figure 3 Electroesophagogram of a patient with score 2 gastroesophageal reflux disease exhibiting slow waves with irregular rhythm and varying frequency, amplitude and conduction velocity from the same electrode and between the 2 electrodes of the same subject. Figure 4 Electroesophagogram of a patient with score 3 gastroesophageal reflux disease recording no electric activity: a "silent" electroesophagogram. Figure 5 Electroesophagogram of a patient with score 3 gastroesophageal reflux disease recording occasional slow waves with no action potentials.
Electroesophagogram in GERD patients
In score 1 GERD, the electric waves' variables were similar to those of the healthy volunteers in all the subjects (p > 0.05, fig  2 ). The SWs were monophasic and negatively deflected and had a regular rhythm (fig  2 ). The frequency, amplitude and conduction velocity are shown in table  1 . Action potentials were randomly recorded following or superimposed over the SWs. This electromyographic pattern was similar from the 2 recording electrodes of the individual subject and was reproducible during the recording period.Score 2 GERD patients exhibited a different electromyographic pattern. The SWs had an irregular rhythm with varying but lower frequency, amplitude and conduction velocity compared to the normal controls (fig.  3 ). The APs occurred randomly and were less frequent than in the normal recordings (fig.  3 ). The SWs and APs differed from one electrode to the other of the same subject and were variable during the recording period. In 8 score 3 GERD patients, the electrodes did not register electric waves; neither SWs nor APs were recorded. A "silent" electromyographic pattern was registered (fig.  4 ); this picture was reproducible during the recording period. In the remaining 2 patients; occasional SWs were recorded that were inconsistent and different from the 2 electrodes of the same patient (fig.  5 ); no APs were recorded at any time during the recording period (fig.  5 ).Discussion The current study has demonstrated that the esophagus possesses an electric activity in the form of regular SWs and APs. The waves in the healthy volunteers were reproducible with identical frequency, amplitude and conduction velocity in the same subject. Previous studies have shown that the APs were coupled with increased esophageal pressure, while the SWs were not [ 12 , 13 ], these findings presumably denote that the APs have a contractile activity [ 12 , 13 ]. Effective esophageal motility is a critical determinant for esophageal clearance of refluxed gastric contents [ 15 ]. A single normal peristaltic wave completely clears the entire barium bolus from the esophagus. If a peristaltic wave fails due to motile dysfunction, there is little or no volume clearance [ 16 ]. Increased exposure of the esophageal mucosa to gastric contents may result from abnormal esophageal motility. Thus, while a defective gastroesophageal barrier accounts for an increased number of gastroesophageal reflux episodes, abnormal esophageal peristalsis results in impaired esophageal acid clearance [ 17 , 18 ]. The relationship between esophageal peristalsis and gastroesophageal reflux has been studied using stationary and ambulatory prolonged esophageal manometry. Various motor-disorders have been detected in GERD including a significantly high rate of incomplete primary peristalsis, changes in esophageal motility and an increased number of non-transmitted contractions [ 6 , 19 ]. Contractions had a shorter duration and a slower propagation velocity [ 20 ]. Patients with abnormal GER but mild esophagitis, or none, had normal amplitude of contractions with increased prevalence of simultaneous contractions [ 16 , 21 ]. Meanwhile patients with severe esophagitis had reduced amplitude of contractions, slow propagation velocities and an increased rate of failed primary peristalsis [ 21 ]. The current study may shed some light on the mechanism of these motor changes in GERD. The electric activity in GERD exhibited different patterns depending on the stage of the GERD. In score 1 GERD, an electroesophagram similar to that of healthy volunteers was recorded. This apparently denotes that the motile activity of the esophagus in score 1 is normal and that the esophageal peristaltic activity can probably clear the esophagus of the refluxed gastric contents. The irregular and diminished esophageal electric wave variables displayed in score 2 GERD are presumably indicative of diminished motile activity and peristalsis of the esophagus with a resulting inhibited reflux clearance rate. The failure of adequate esophageal clearance is probably responsible for the clinical manifestations and investigative results encountered in score 2 GERD. With progress of the condition to score 3, there is probably no motor or peristaltic esophageal activity as evidenced by the absence of the esophageal electric waves. In such case, we presume that there is no esophageal clearance. We do not know the cause of the diminished esophageal electric activity in GERD. Is it due to the refluxed acid material or to the resulting esophageal inflammation? It may be argued that the refluxed acid content into the esophagus inhibits its motile and peristaltic activity. However, the current and earlier studies have demonstrated normal peristalsis in score 1 GERD in which acid reflux was manifest [ 16 ]. Furthermore, the current study showed normal EMG activity in this condition. Probably these findings negate the role of acid reflux as inhibitor of the peristaltic and electric activity in GERD. What then could be the cause of the deranged electric activity and peristaltic movement in the more advanced stages of GERD? A new theory of the pathogenesis of diminished electric activity in GERD The electric waves seem to be generated from the interstitial cells of Cajal that are located at the level of the myenteric plexus and in the circular muscle layer of the esophageal wall [ 22 - 24 ]. They are considered as the generators of the spontaneous pacemaker activity in the smooth muscle layers of the gut [ 22 - 24 ] and are also involved in neurotransmission [ 25 - 27 ]. They mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium. In the advanced stages of GERD it may be assumed that the inflammatory changes in the esophageal wall have involved the interstitial cells of Cajal. Destruction of these cells appears to occur in grades that are in accordance with the GERD socres. It seems that in score 1 GERD, the mucosal inflammatory changes of the esophagus, if present, have not involved the Cajal cells yet. With the more advanced stages of the disease as in scores 2 and 3, the Cajal cells are presumingly being gradually destroyed by the advancing esophageal inflammatory process. The diminished SW variables encountered in score 2 GERD seem to be due to partial involvement of Cajal cells in the inflammatory process; the cells are not yet completely destroyed and are still mediating electric and peristaltic activity. However the Cajal cells in score 3 GERD are believed to be extremely injured so that they cannot generate electric activity. Diagnostic role of electroesophagogram in GERD There are various methods for the diagnosis of GERD. They include pressure measurements using water-perfused manometry catheters, external transducers or intraluminal transducers, pH-metry and others [ 28 , 29 ]. However they might have disadvantages [ 30 ]. In view of the results of our above study, the introduction of the electroesophagogram as an investigative tool may be a valuable addition to the armamentarium of esophageal investigations of GERD.
Discussion
The current study has demonstrated that the esophagus possesses an electric activity in the form of regular SWs and APs. The waves in the healthy volunteers were reproducible with identical frequency, amplitude and conduction velocity in the same subject. Previous studies have shown that the APs were coupled with increased esophageal pressure, while the SWs were not [ 12 , 13 ], these findings presumably denote that the APs have a contractile activity [ 12 , 13 ].Effective esophageal motility is a critical determinant for esophageal clearance of refluxed gastric contents [ 15 ]. A single normal peristaltic wave completely clears the entire barium bolus from the esophagus. If a peristaltic wave fails due to motile dysfunction, there is little or no volume clearance [ 16 ]. Increased exposure of the esophageal mucosa to gastric contents may result from abnormal esophageal motility. Thus, while a defective gastroesophageal barrier accounts for an increased number of gastroesophageal reflux episodes, abnormal esophageal peristalsis results in impaired esophageal acid clearance [ 17 , 18 ].The relationship between esophageal peristalsis and gastroesophageal reflux has been studied using stationary and ambulatory prolonged esophageal manometry. Various motor-disorders have been detected in GERD including a significantly high rate of incomplete primary peristalsis, changes in esophageal motility and an increased number of non-transmitted contractions [ 6 , 19 ]. Contractions had a shorter duration and a slower propagation velocity [ 20 ].Patients with abnormal GER but mild esophagitis, or none, had normal amplitude of contractions with increased prevalence of simultaneous contractions [ 16 , 21 ]. Meanwhile patients with severe esophagitis had reduced amplitude of contractions, slow propagation velocities and an increased rate of failed primary peristalsis [ 21 ]. The current study may shed some light on the mechanism of these motor changes in GERD.The electric activity in GERD exhibited different patterns depending on the stage of the GERD. In score 1 GERD, an electroesophagram similar to that of healthy volunteers was recorded. This apparently denotes that the motile activity of the esophagus in score 1 is normal and that the esophageal peristaltic activity can probably clear the esophagus of the refluxed gastric contents. The irregular and diminished esophageal electric wave variables displayed in score 2 GERD are presumably indicative of diminished motile activity and peristalsis of the esophagus with a resulting inhibited reflux clearance rate. The failure of adequate esophageal clearance is probably responsible for the clinical manifestations and investigative results encountered in score 2 GERD. With progress of the condition to score 3, there is probably no motor or peristaltic esophageal activity as evidenced by the absence of the esophageal electric waves. In such case, we presume that there is no esophageal clearance.We do not know the cause of the diminished esophageal electric activity in GERD. Is it due to the refluxed acid material or to the resulting esophageal inflammation? It may be argued that the refluxed acid content into the esophagus inhibits its motile and peristaltic activity. However, the current and earlier studies have demonstrated normal peristalsis in score 1 GERD in which acid reflux was manifest [ 16 ]. Furthermore, the current study showed normal EMG activity in this condition. Probably these findings negate the role of acid reflux as inhibitor of the peristaltic and electric activity in GERD.What then could be the cause of the deranged electric activity and peristaltic movement in the more advanced stages of GERD?A new theory of the pathogenesis of diminished electric activity in GERD The electric waves seem to be generated from the interstitial cells of Cajal that are located at the level of the myenteric plexus and in the circular muscle layer of the esophageal wall [ 22 - 24 ]. They are considered as the generators of the spontaneous pacemaker activity in the smooth muscle layers of the gut [ 22 - 24 ] and are also involved in neurotransmission [ 25 - 27 ]. They mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium. In the advanced stages of GERD it may be assumed that the inflammatory changes in the esophageal wall have involved the interstitial cells of Cajal. Destruction of these cells appears to occur in grades that are in accordance with the GERD socres. It seems that in score 1 GERD, the mucosal inflammatory changes of the esophagus, if present, have not involved the Cajal cells yet. With the more advanced stages of the disease as in scores 2 and 3, the Cajal cells are presumingly being gradually destroyed by the advancing esophageal inflammatory process. The diminished SW variables encountered in score 2 GERD seem to be due to partial involvement of Cajal cells in the inflammatory process; the cells are not yet completely destroyed and are still mediating electric and peristaltic activity. However the Cajal cells in score 3 GERD are believed to be extremely injured so that they cannot generate electric activity.
A new theory of the pathogenesis of diminished electric activity in GERD
The electric waves seem to be generated from the interstitial cells of Cajal that are located at the level of the myenteric plexus and in the circular muscle layer of the esophageal wall [ 22 - 24 ]. They are considered as the generators of the spontaneous pacemaker activity in the smooth muscle layers of the gut [ 22 - 24 ] and are also involved in neurotransmission [ 25 - 27 ]. They mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium.In the advanced stages of GERD it may be assumed that the inflammatory changes in the esophageal wall have involved the interstitial cells of Cajal. Destruction of these cells appears to occur in grades that are in accordance with the GERD socres. It seems that in score 1 GERD, the mucosal inflammatory changes of the esophagus, if present, have not involved the Cajal cells yet. With the more advanced stages of the disease as in scores 2 and 3, the Cajal cells are presumingly being gradually destroyed by the advancing esophageal inflammatory process. The diminished SW variables encountered in score 2 GERD seem to be due to partial involvement of Cajal cells in the inflammatory process; the cells are not yet completely destroyed and are still mediating electric and peristaltic activity. However the Cajal cells in score 3 GERD are believed to be extremely injured so that they cannot generate electric activity.Diagnostic role of electroesophagogram in GERD There are various methods for the diagnosis of GERD. They include pressure measurements using water-perfused manometry catheters, external transducers or intraluminal transducers, pH-metry and others [ 28 , 29 ]. However they might have disadvantages [ 30 ]. In view of the results of our above study, the introduction of the electroesophagogram as an investigative tool may be a valuable addition to the armamentarium of esophageal investigations of GERD.
Diagnostic role of electroesophagogram in GERD
There are various methods for the diagnosis of GERD. They include pressure measurements using water-perfused manometry catheters, external transducers or intraluminal transducers, pH-metry and others [ 28 , 29 ]. However they might have disadvantages [ 30 ]. In view of the results of our above study, the introduction of the electroesophagogram as an investigative tool may be a valuable addition to the armamentarium of esophageal investigations of GERD.Conclusion The electric activity in GERD expressed 3 different patterns depending on the stage of GERD. In score 1 GERD, a normal electroesophagogram was recorded which would denote normal esophageal motile activity and acid clearance. Score 2 GERD exhibited irregular and diminished esophageal electric waves' variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" electroesophagogram was recorded with probably no acid clearance. A new theory of the pathogenesis of diminished electric activity in GERD is put forward. It is postulated that the interstitial cells of Cajal which are considered as the generators of the pacemaker activity and electric waves, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The electroesophagogram may serve as an investigative tool in diagnosing the various GERD stages, especially if they can be recorded percutaneously.
Conclusion
The electric activity in GERD expressed 3 different patterns depending on the stage of GERD. In score 1 GERD, a normal electroesophagogram was recorded which would denote normal esophageal motile activity and acid clearance. Score 2 GERD exhibited irregular and diminished esophageal electric waves' variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" electroesophagogram was recorded with probably no acid clearance. A new theory of the pathogenesis of diminished electric activity in GERD is put forward. It is postulated that the interstitial cells of Cajal which are considered as the generators of the pacemaker activity and electric waves, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The electroesophagogram may serve as an investigative tool in diagnosing the various GERD stages, especially if they can be recorded percutaneously.List of abbreviations gastroesophageal reflux disease = GERD (electroesophagogram = EEG slow waves = SWs action potentials = APs standard deviation = SD
List of abbreviations
gastroesophageal reflux disease = GERD(electroesophagogram = EEGslow waves = SWsaction potentials = APsstandard deviation = SDAuthors contributions AS: Study design/ planning OES: Data collection/entry, Data analysis/statistics, Literature analysis/search IS: Data collection/entry, Data analysis/statistics, Literature analysis/search AS: Data collection/entry, Preparation manuscript
Authors contributions
AS: Study design/ planningOES: Data collection/entry, Data analysis/statistics, Literature analysis/searchIS: Data collection/entry, Data analysis/statistics, Literature analysis/searchAS: Data collection/entry, Preparation manuscriptPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy.
Background
Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy.Methods Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry.
Methods
Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry.Results Seventy-six  infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants,  p < 0.001 . The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants,  p < 0.001 . The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%.
Results
Seventy-six  infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants,  p < 0.001 . The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants,  p < 0.001 . The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%.Seventy-sixSeventy-sixp < 0.001p < 0.001Conclusion The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy.
Conclusion
The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy.

Background Endemic HIV infection, in sub-Saharan Africa, where in many countries more than 20% of pregnant women are HIV seropositive leads to a diagnostic problem in the evaluation of their infants. Without intervention, more than 25% of infants born to seropositive women will acquire HIV infection in the first year of life. HIV testing with enzyme immunoassay-based rapid tests have expanded capacity to identify seropositive women and provide interventions, but even with single dose Nevirapine and other antiretrovirals, infection of infants still exceeds 10% in the first year of life. Serologic tests for HIV do not accurately identify those infants who have acquired infection within the first 18 months of life because of transplacentally acquired maternal IgG antibodies. As antimicrobial and antiviral interventions are developed to reduce morbidity and mortality, among infants born to seropositive women, the early diagnosis of infection is increasingly important. The gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR [ 1 - 3 ]. With the increasing availability of single dose nevirapine for prevention of mother-to-child transmission (MTCT) of HIV [ 4 ] and generic antiretroviral drugs for treatment of AIDS in resource-poor countries, there is an urgent need to develop alternative laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infants who meet the criteria for commencing cotrimoxazole prophylaxis and/or initiation of ARV therapy. The World Health Organization (WHO) recommends initiation of highly active antiretroviral therapy (HAART) in HIV-seropositive infants under the age of 18 months, who have WHO Pediatric Stage III disease and CD4 percentage (%CD4) <20% in resource-poor countries where %CD4 are available but virologic tests (DNA PCR, RT-PCR or immune-complex dissociated p24 antigen) for confirmation of HIV infection are not available [ 5 ]. Thus, in infants under the age of 18 months, a single laboratory test that can identify both HIV-infection and provide %CD4, two important parameters for decision-making in initiating HAART, may be useful. Infants who are positively identified as HIV-infected and meet clinical criteria are likely to benefit from HAART. Immunological changes in HIV-1 infection include a decrease in CD4 +  cells, a transient increase in CD8 +  cells, total lymphocytes and inversion of the CD4/CD8 ratio [ 6 , 7 ]. As HIV infection progresses, the CD4 +  cells decline, while the CD8 +  cells which may remain at high levels for long periods, eventually decrease but not to baseline levels. Since in healthy children the CD4 +  and CD8 +  cells account for 60% and 30% of the T lymphocytes respectively, a normal CD4/CD8 ratio should always be >1.0. Thus, in HIV-1 infection where there is a decrease in CD4 +  cells and an increase in CD8 +  cells, the reversal of the CD4/CD8 to <1.0 should in theory be useful for diagnosis of HIV-1 infection. Certain flow cytometers when used in tandem with a haematological analyzer can provide absolute CD4 +  cell counts, their percentages as well as the CD4/CD8 ratio. The use of the ratio in combination with %CD4, may lead to a timely identification of infected infants, who meet the WHO criteria for initiation of CTX prophylaxis and/or ARV therapy. While the infrastructure needed to conduct flow-cytometric analyses of HIV infection are still largely confined to a few centers, an increasing number of point of care diagnostic testing systems, inexpensive methods to measure CD4 cells are currently in development. These include the development and evaluation of simplified volumetric flow cytometric methods using a low cost flow cytometer that can be powered from a car battery or by solar panels (Cyflow SL, Partec, Munster, Germany) by Cassens and colleagues [ 8 , 9 ] and modification of a commercially available 4-parameter flow cytometer, Luminex 100 (Luminex, Austin Texas, USA) to a compact portable prototype instrument that can operate with a 12-volt rechargeable battery [ 10 ]. Furthermore, use of generic CD4, CD8 and CD45 fluorescence-conjugated monoclonal antibodies can reduce the cost of determining T cell subset profile even when employing standard flow cytometers [ 10 , 11 ]. The main objective of the current study was to evaluate the CD4/CD8 ratio for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years among infants where DNA PCR was performed to diagnose HIV infection.
Background
Endemic HIV infection, in sub-Saharan Africa, where in many countries more than 20% of pregnant women are HIV seropositive leads to a diagnostic problem in the evaluation of their infants. Without intervention, more than 25% of infants born to seropositive women will acquire HIV infection in the first year of life. HIV testing with enzyme immunoassay-based rapid tests have expanded capacity to identify seropositive women and provide interventions, but even with single dose Nevirapine and other antiretrovirals, infection of infants still exceeds 10% in the first year of life. Serologic tests for HIV do not accurately identify those infants who have acquired infection within the first 18 months of life because of transplacentally acquired maternal IgG antibodies. As antimicrobial and antiviral interventions are developed to reduce morbidity and mortality, among infants born to seropositive women, the early diagnosis of infection is increasingly important. The gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR [ 1 - 3 ]. With the increasing availability of single dose nevirapine for prevention of mother-to-child transmission (MTCT) of HIV [ 4 ] and generic antiretroviral drugs for treatment of AIDS in resource-poor countries, there is an urgent need to develop alternative laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infants who meet the criteria for commencing cotrimoxazole prophylaxis and/or initiation of ARV therapy.The World Health Organization (WHO) recommends initiation of highly active antiretroviral therapy (HAART) in HIV-seropositive infants under the age of 18 months, who have WHO Pediatric Stage III disease and CD4 percentage (%CD4) <20% in resource-poor countries where %CD4 are available but virologic tests (DNA PCR, RT-PCR or immune-complex dissociated p24 antigen) for confirmation of HIV infection are not available [ 5 ]. Thus, in infants under the age of 18 months, a single laboratory test that can identify both HIV-infection and provide %CD4, two important parameters for decision-making in initiating HAART, may be useful. Infants who are positively identified as HIV-infected and meet clinical criteria are likely to benefit from HAART.Immunological changes in HIV-1 infection include a decrease in CD4 +  cells, a transient increase in CD8 +  cells, total lymphocytes and inversion of the CD4/CD8 ratio [ 6 , 7 ]. As HIV infection progresses, the CD4 +  cells decline, while the CD8 +  cells which may remain at high levels for long periods, eventually decrease but not to baseline levels. Since in healthy children the CD4 +  and CD8 +  cells account for 60% and 30% of the T lymphocytes respectively, a normal CD4/CD8 ratio should always be >1.0. Thus, in HIV-1 infection where there is a decrease in CD4 +  cells and an increase in CD8 +  cells, the reversal of the CD4/CD8 to <1.0 should in theory be useful for diagnosis of HIV-1 infection.Certain flow cytometers when used in tandem with a haematological analyzer can provide absolute CD4 +  cell counts, their percentages as well as the CD4/CD8 ratio. The use of the ratio in combination with %CD4, may lead to a timely identification of infected infants, who meet the WHO criteria for initiation of CTX prophylaxis and/or ARV therapy. While the infrastructure needed to conduct flow-cytometric analyses of HIV infection are still largely confined to a few centers, an increasing number of point of care diagnostic testing systems, inexpensive methods to measure CD4 cells are currently in development. These include the development and evaluation of simplified volumetric flow cytometric methods using a low cost flow cytometer that can be powered from a car battery or by solar panels (Cyflow SL, Partec, Munster, Germany) by Cassens and colleagues [ 8 , 9 ] and modification of a commercially available 4-parameter flow cytometer, Luminex 100 (Luminex, Austin Texas, USA) to a compact portable prototype instrument that can operate with a 12-volt rechargeable battery [ 10 ]. Furthermore, use of generic CD4, CD8 and CD45 fluorescence-conjugated monoclonal antibodies can reduce the cost of determining T cell subset profile even when employing standard flow cytometers [ 10 , 11 ].The main objective of the current study was to evaluate the CD4/CD8 ratio for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years among infants where DNA PCR was performed to diagnose HIV infection.Materials and Methods Study cohort The infant specimens used in this study were obtained from two independent prospective studies; the short course zidovudine (AZT) and Pediatric AIDS Clinical definition (PACD). The Medical Research Council and the Institutional Ethics Committees approved both studies and present study. The infants included in the study were under the age of 2 years. All the infants were breastfeeding at the time of specimen collection. Infants who were followed in the short course AZT study aimed at preventing MTCT were enrolled between May 2001 and June 2002. In the AZT study, mothers received short course AZT starting at 36 weeks gestation and throughout labor. Their infants received AZT for 7 days. In the PACD study, hospitalized children aged between 2 months and 18 months were prospectively enrolled into the study, between July 2002 and July 2003, following informed consent from their mothers. The mothers of these children did not receive antiretroviral therapy for prevention of MTCT nor for HIV disease. The PACD study is a hospital based analytical cross-sectional survey. Laboratory specimens were obtained once from each subject. Children presenting in the moribund state, requiring immediate resuscitation or those with known HIV status or those whose mothers/guardians refused to sign the informed consent were excluded from the study. The objective of the PACD study is to identify clinical symptoms, associated with HIV-1 infection in infants under the age of 18 months, which may be used in the absence of laboratory tests, for HIV-1 diagnosis. Blood collection and processing A total volume of 2 ml whole blood was collected in ethylenediamine tetraacetic acid from each of the 137 infants in the short course AZT and the PACD studies. The whole blood was then aliquoted into two tubes (500 microlitres in each) for determination of T cell subset profiles, and the second for DNA PCR and the rest centrifuged at 200 g to obtain plasma, which was stored at -80°C. The laboratory tests described below were conducted in a blind fashion. Flow cytometry analysis T cell subset profiles were determined by flow cytometry using a Coulter Epics XL equipped with System II software (Beckman Coulter, Miami, Florida, USA) within 4 hours of blood collection. This flow cytometer was run, in a double platform setting where the absolute counts for both white blood cells and lymphocytes were obtained on a Celldyn 3500R haematological analyzer (Abbott, GmbH, Germany). Then from the combined results, the absolute CD4 +  and CD8 +  cell counts, CD4/CD8 ratios, as well as the %CD4 and %CD8 values among lymphocytes were automatically calculated. DNA PCR analysis DNA PCR Roche amplification assay version 1.5 (Roche Diagnostics, Branchburg, NJ, USA) was employed as the reference standard for diagnosis of infant HIV-1 infection status. DNA extraction, amplification and detection were performed and results interpreted following the manufacturer's instructions (Roche Diagnostics, Branchburg, NJ, USA) as we previously described [ 12 , 13 ]. Infant grouping Reference ranges of T cell subset profiles for infants and children are usually stratified by age as <12 months, 1 to 5 years and 6–12 years. In the current study, in addition to overall evaluation of samples obtained from all the infants aged 0 to 18 months, the evaluated parameters were also compared based on infant age groups 0–11, and 12–18 months. Statistical Analysis Diagnostic tests (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and test efficiency (TE) with 95% confidence interval) were used to assess the assay under evaluation with DNA PCR as the reference standard. A MTCT prevalence rate of 30.7% (based on the current rate in Zimbabwe) was used in the calculations of PPV and NPV. Sensitivity was defined as the percentage of reference standard HIV positive samples found reactive by assay under evaluation. Specificity was defined as the percentage of reference standard HIV-negative samples that were negative by the assay under evaluation. TE refers to the overall ability of a test to correctly identify all positives and negatives (the absence of false positives and false negatives). It is a combination of the sensitivity and the specificity of an assay and gives an idea of the total effectiveness of the assay. PPV was defined as the probability that a specimen had CD4:CD8 ratio less than 1 when the test was DNA PCR positive. NPV was defined as the probability that a specimen does not have a CD4:CD8 ratio ≥ 1 when the test was DNA PCR negative. Comparisons of T cells between infected and uninfected infants were done using non-parametic equivalent of the T-test (Kruskal-Wallis test).  P  values less than 0.05 were considered statistically significant.
Materials and Methods
Study cohort The infant specimens used in this study were obtained from two independent prospective studies; the short course zidovudine (AZT) and Pediatric AIDS Clinical definition (PACD). The Medical Research Council and the Institutional Ethics Committees approved both studies and present study. The infants included in the study were under the age of 2 years. All the infants were breastfeeding at the time of specimen collection. Infants who were followed in the short course AZT study aimed at preventing MTCT were enrolled between May 2001 and June 2002. In the AZT study, mothers received short course AZT starting at 36 weeks gestation and throughout labor. Their infants received AZT for 7 days. In the PACD study, hospitalized children aged between 2 months and 18 months were prospectively enrolled into the study, between July 2002 and July 2003, following informed consent from their mothers. The mothers of these children did not receive antiretroviral therapy for prevention of MTCT nor for HIV disease. The PACD study is a hospital based analytical cross-sectional survey. Laboratory specimens were obtained once from each subject. Children presenting in the moribund state, requiring immediate resuscitation or those with known HIV status or those whose mothers/guardians refused to sign the informed consent were excluded from the study. The objective of the PACD study is to identify clinical symptoms, associated with HIV-1 infection in infants under the age of 18 months, which may be used in the absence of laboratory tests, for HIV-1 diagnosis.
Study cohort
The infant specimens used in this study were obtained from two independent prospective studies; the short course zidovudine (AZT) and Pediatric AIDS Clinical definition (PACD). The Medical Research Council and the Institutional Ethics Committees approved both studies and present study. The infants included in the study were under the age of 2 years. All the infants were breastfeeding at the time of specimen collection.Infants who were followed in the short course AZT study aimed at preventing MTCT were enrolled between May 2001 and June 2002. In the AZT study, mothers received short course AZT starting at 36 weeks gestation and throughout labor. Their infants received AZT for 7 days.In the PACD study, hospitalized children aged between 2 months and 18 months were prospectively enrolled into the study, between July 2002 and July 2003, following informed consent from their mothers. The mothers of these children did not receive antiretroviral therapy for prevention of MTCT nor for HIV disease. The PACD study is a hospital based analytical cross-sectional survey. Laboratory specimens were obtained once from each subject. Children presenting in the moribund state, requiring immediate resuscitation or those with known HIV status or those whose mothers/guardians refused to sign the informed consent were excluded from the study. The objective of the PACD study is to identify clinical symptoms, associated with HIV-1 infection in infants under the age of 18 months, which may be used in the absence of laboratory tests, for HIV-1 diagnosis.Blood collection and processing A total volume of 2 ml whole blood was collected in ethylenediamine tetraacetic acid from each of the 137 infants in the short course AZT and the PACD studies. The whole blood was then aliquoted into two tubes (500 microlitres in each) for determination of T cell subset profiles, and the second for DNA PCR and the rest centrifuged at 200 g to obtain plasma, which was stored at -80°C. The laboratory tests described below were conducted in a blind fashion.
Blood collection and processing
A total volume of 2 ml whole blood was collected in ethylenediamine tetraacetic acid from each of the 137 infants in the short course AZT and the PACD studies. The whole blood was then aliquoted into two tubes (500 microlitres in each) for determination of T cell subset profiles, and the second for DNA PCR and the rest centrifuged at 200 g to obtain plasma, which was stored at -80°C.The laboratory tests described below were conducted in a blind fashion.Flow cytometry analysis T cell subset profiles were determined by flow cytometry using a Coulter Epics XL equipped with System II software (Beckman Coulter, Miami, Florida, USA) within 4 hours of blood collection. This flow cytometer was run, in a double platform setting where the absolute counts for both white blood cells and lymphocytes were obtained on a Celldyn 3500R haematological analyzer (Abbott, GmbH, Germany). Then from the combined results, the absolute CD4 +  and CD8 +  cell counts, CD4/CD8 ratios, as well as the %CD4 and %CD8 values among lymphocytes were automatically calculated.
Flow cytometry analysis
T cell subset profiles were determined by flow cytometry using a Coulter Epics XL equipped with System II software (Beckman Coulter, Miami, Florida, USA) within 4 hours of blood collection. This flow cytometer was run, in a double platform setting where the absolute counts for both white blood cells and lymphocytes were obtained on a Celldyn 3500R haematological analyzer (Abbott, GmbH, Germany). Then from the combined results, the absolute CD4 +  and CD8 +  cell counts, CD4/CD8 ratios, as well as the %CD4 and %CD8 values among lymphocytes were automatically calculated.DNA PCR analysis DNA PCR Roche amplification assay version 1.5 (Roche Diagnostics, Branchburg, NJ, USA) was employed as the reference standard for diagnosis of infant HIV-1 infection status. DNA extraction, amplification and detection were performed and results interpreted following the manufacturer's instructions (Roche Diagnostics, Branchburg, NJ, USA) as we previously described [ 12 , 13 ].
DNA PCR analysis
DNA PCR Roche amplification assay version 1.5 (Roche Diagnostics, Branchburg, NJ, USA) was employed as the reference standard for diagnosis of infant HIV-1 infection status. DNA extraction, amplification and detection were performed and results interpreted following the manufacturer's instructions (Roche Diagnostics, Branchburg, NJ, USA) as we previously described [ 12 , 13 ].Infant grouping Reference ranges of T cell subset profiles for infants and children are usually stratified by age as <12 months, 1 to 5 years and 6–12 years. In the current study, in addition to overall evaluation of samples obtained from all the infants aged 0 to 18 months, the evaluated parameters were also compared based on infant age groups 0–11, and 12–18 months.
Infant grouping
Reference ranges of T cell subset profiles for infants and children are usually stratified by age as <12 months, 1 to 5 years and 6–12 years. In the current study, in addition to overall evaluation of samples obtained from all the infants aged 0 to 18 months, the evaluated parameters were also compared based on infant age groups 0–11, and 12–18 months.Statistical Analysis Diagnostic tests (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and test efficiency (TE) with 95% confidence interval) were used to assess the assay under evaluation with DNA PCR as the reference standard. A MTCT prevalence rate of 30.7% (based on the current rate in Zimbabwe) was used in the calculations of PPV and NPV. Sensitivity was defined as the percentage of reference standard HIV positive samples found reactive by assay under evaluation. Specificity was defined as the percentage of reference standard HIV-negative samples that were negative by the assay under evaluation. TE refers to the overall ability of a test to correctly identify all positives and negatives (the absence of false positives and false negatives). It is a combination of the sensitivity and the specificity of an assay and gives an idea of the total effectiveness of the assay. PPV was defined as the probability that a specimen had CD4:CD8 ratio less than 1 when the test was DNA PCR positive. NPV was defined as the probability that a specimen does not have a CD4:CD8 ratio ≥ 1 when the test was DNA PCR negative. Comparisons of T cells between infected and uninfected infants were done using non-parametic equivalent of the T-test (Kruskal-Wallis test).  P  values less than 0.05 were considered statistically significant.
Statistical Analysis
Diagnostic tests (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and test efficiency (TE) with 95% confidence interval) were used to assess the assay under evaluation with DNA PCR as the reference standard. A MTCT prevalence rate of 30.7% (based on the current rate in Zimbabwe) was used in the calculations of PPV and NPV. Sensitivity was defined as the percentage of reference standard HIV positive samples found reactive by assay under evaluation. Specificity was defined as the percentage of reference standard HIV-negative samples that were negative by the assay under evaluation. TE refers to the overall ability of a test to correctly identify all positives and negatives (the absence of false positives and false negatives). It is a combination of the sensitivity and the specificity of an assay and gives an idea of the total effectiveness of the assay. PPV was defined as the probability that a specimen had CD4:CD8 ratio less than 1 when the test was DNA PCR positive. NPV was defined as the probability that a specimen does not have a CD4:CD8 ratio ≥ 1 when the test was DNA PCR negative.Comparisons of T cells between infected and uninfected infants were done using non-parametic equivalent of the T-test (Kruskal-Wallis test).  P  values less than 0.05 were considered statistically significant.PResults Of the 137 infant specimens tested using DNA PCR, 76 were HIV-1 positive and 61 were HIV-1 negative. The 76 PCR positive infants included 9 infants who had evidence of  in utero  transmission as determined by serial DNA PCR of birth and subsequent samples tested in longitudinal short course AZT MTCT studies (Zijenah et al, unpublished data). T cell subset of infected and uninfected infants T lymphocyte subset profiles were performed for the 137 infants who had whole blood specimens for flow cytometry. The median age of these infants at specimen collection was 5.5 months (Interquartile Range [IQR]: 3–13) and 8.0 months (IQR: 4–14) for HIV infected and uninfected respectively ( p = 0.08 ). As expected, the median CD4 +  cell counts of PCR negative infants were significantly higher than those of the PCR positive infants,  p < 0.001  (Table  1 ). Inversely, the median CD8 +  cell counts were significantly higher among PCR positive infants than PCR negative infants,  p < 0.001  (Table  1 ). The median CD4/CD8 ratio of the PCR positive infants (0.4, IQR: 0.3–0.6) was significantly lower than that of the PCR negative infants (1.8, IQR: 1.4–2.3),  p < 0.001 . The median %CD4 of PCR positive infants was also significantly lower than that of PCR negative infants,  p = < 0.01  (Table  1 ). Table 1 T cell subset profile of HIV-1 infected and uninfected infants PCR Positive (n = 76) PCR Negative (n = 61) P  value a Median age (months) 5.5 (IQR: 3–13) 8 (IQR: 4–14) 0.08 Median CD4 + (cells/μL) 521.5 (IQR: 323–805) 1356 (IQR: 916–1769) <0.001 Median CD8 + (cells/μL) 1302.5 (IQR: 829–2054) 799 (IQR: 471–1020) <0.001 Median CD4/CD8 ratio 0.4 (IQR: 0.3–0.6) 1.8 (IQR: 1.4–2.3) <0.001 Median %CD4 13.9 (IQR: 9.2–19.1) 29.9 (IQR: 25.3–34.5) <0.001 Median %CD8 31.3 (IQR: 22.3–42.9) 18.4 (IQR: 14.2–21.5) <0.001 Abbreviations: PCR, polymerase chain reaction; n, number tested;  P  value a  for statistical significance between group medians was estimated using the Kruskal-Wallis test. DNA PCR versus CD4/CD8 ratio Seventy-five infants of the 76 PCR positive group (98.7%) had CD4/CD8 ratio <1 while 60 infants of the 61 who were PCR negative had CD4/CD8 ratio ≥ 1. All the 9 infants infected in utero had a CD4:CD8 ratio less than 1. Their median CD4/CD8 ratio was similar to that of the rest of the PCR positive babies (0.4; IQR: 0.3–0.6), albeit the numbers are too small for statistical significance considerations. The infant (aged 11 months at specimen collection) who was DNA PCR positive with a CD4/CD8 ratio of 1.1 was from the PACD study. The PCR negative infant (aged 4 months at specimen collection) with a CD4/CD8 ratio of 0.3 was also from the PACD cohort. The overall sensitivity and specificity of the CD4/CD8 ratio were 98.7% (95% CI: 96.1–100 and 98.3% (95% Confidence Interval (CI): 95.1–100) respectively with PPV and NPV of 96.3% and 99.4% respectively and a test efficiency of 98.5% (95% CI: 96.5–100) (Table  2 ). Comparison of all the evaluated parameters between the 0–11 and 12–18 months infant age groups showed that the 95% CI overlap between the groups which implies no statistically significant difference in these two age groups. Table 2 Evaluated parameters for CD4/CD8 ratio for the three infant age groups using DNA PCR as reference standard 0–18 months infant age group (n = 137) 0–11 months infant age group (n = 95) 12–18 months infant age group (n = 42) %Sensitivity 98.7 (CI: 96.1–100) 98.2 (CI: 94.7–100) 100 (CI: 100–100) %Specificity 98.4 (CI: 95.2–100) 97.5 (CI: 92.7–100) 100 (CI: 100–100) %PPV 96.4 94.6 100 %NPV 99.4 99.2 100 %TE 98.5 (CI: 96.5–100) 97.9 (CI: 95.0–100) 100 (CI: 100–100) Abbreviations: n, number tested; NPV, negative predictive value; PPV, positive predictive value; TE, test efficiency.
Results
Of the 137 infant specimens tested using DNA PCR, 76 were HIV-1 positive and 61 were HIV-1 negative. The 76 PCR positive infants included 9 infants who had evidence of  in utero  transmission as determined by serial DNA PCR of birth and subsequent samples tested in longitudinal short course AZT MTCT studies (Zijenah et al, unpublished data).in uteroT cell subset of infected and uninfected infants T lymphocyte subset profiles were performed for the 137 infants who had whole blood specimens for flow cytometry. The median age of these infants at specimen collection was 5.5 months (Interquartile Range [IQR]: 3–13) and 8.0 months (IQR: 4–14) for HIV infected and uninfected respectively ( p = 0.08 ). As expected, the median CD4 +  cell counts of PCR negative infants were significantly higher than those of the PCR positive infants,  p < 0.001  (Table  1 ). Inversely, the median CD8 +  cell counts were significantly higher among PCR positive infants than PCR negative infants,  p < 0.001  (Table  1 ). The median CD4/CD8 ratio of the PCR positive infants (0.4, IQR: 0.3–0.6) was significantly lower than that of the PCR negative infants (1.8, IQR: 1.4–2.3),  p < 0.001 . The median %CD4 of PCR positive infants was also significantly lower than that of PCR negative infants,  p = < 0.01  (Table  1 ). Table 1 T cell subset profile of HIV-1 infected and uninfected infants PCR Positive (n = 76) PCR Negative (n = 61) P  value a Median age (months) 5.5 (IQR: 3–13) 8 (IQR: 4–14) 0.08 Median CD4 + (cells/μL) 521.5 (IQR: 323–805) 1356 (IQR: 916–1769) <0.001 Median CD8 + (cells/μL) 1302.5 (IQR: 829–2054) 799 (IQR: 471–1020) <0.001 Median CD4/CD8 ratio 0.4 (IQR: 0.3–0.6) 1.8 (IQR: 1.4–2.3) <0.001 Median %CD4 13.9 (IQR: 9.2–19.1) 29.9 (IQR: 25.3–34.5) <0.001 Median %CD8 31.3 (IQR: 22.3–42.9) 18.4 (IQR: 14.2–21.5) <0.001 Abbreviations: PCR, polymerase chain reaction; n, number tested;  P  value a  for statistical significance between group medians was estimated using the Kruskal-Wallis test.
T cell subset of infected and uninfected infants
T lymphocyte subset profiles were performed for the 137 infants who had whole blood specimens for flow cytometry. The median age of these infants at specimen collection was 5.5 months (Interquartile Range [IQR]: 3–13) and 8.0 months (IQR: 4–14) for HIV infected and uninfected respectively ( p = 0.08 ). As expected, the median CD4 +  cell counts of PCR negative infants were significantly higher than those of the PCR positive infants,  p < 0.001  (Table  1 ). Inversely, the median CD8 +  cell counts were significantly higher among PCR positive infants than PCR negative infants,  p < 0.001  (Table  1 ). The median CD4/CD8 ratio of the PCR positive infants (0.4, IQR: 0.3–0.6) was significantly lower than that of the PCR negative infants (1.8, IQR: 1.4–2.3),  p < 0.001 . The median %CD4 of PCR positive infants was also significantly lower than that of PCR negative infants,  p = < 0.01  (Table  1 ).p = 0.08p < 0.001p < 0.001p < 0.001p = < 0.01T cell subset profile of HIV-1 infected and uninfected infantsPCR Positive (n = 76)PCR Negative (n = 61)P  value aPMedian age (months)Median CD4 + (cells/μL)Median CD8 + (cells/μL)Median CD4/CD8 ratioMedian %CD4Median %CD8Abbreviations: PCR, polymerase chain reaction; n, number tested;  P  value a  for statistical significance between group medians was estimated using the Kruskal-Wallis test.PDNA PCR versus CD4/CD8 ratio Seventy-five infants of the 76 PCR positive group (98.7%) had CD4/CD8 ratio <1 while 60 infants of the 61 who were PCR negative had CD4/CD8 ratio ≥ 1. All the 9 infants infected in utero had a CD4:CD8 ratio less than 1. Their median CD4/CD8 ratio was similar to that of the rest of the PCR positive babies (0.4; IQR: 0.3–0.6), albeit the numbers are too small for statistical significance considerations. The infant (aged 11 months at specimen collection) who was DNA PCR positive with a CD4/CD8 ratio of 1.1 was from the PACD study. The PCR negative infant (aged 4 months at specimen collection) with a CD4/CD8 ratio of 0.3 was also from the PACD cohort. The overall sensitivity and specificity of the CD4/CD8 ratio were 98.7% (95% CI: 96.1–100 and 98.3% (95% Confidence Interval (CI): 95.1–100) respectively with PPV and NPV of 96.3% and 99.4% respectively and a test efficiency of 98.5% (95% CI: 96.5–100) (Table  2 ). Comparison of all the evaluated parameters between the 0–11 and 12–18 months infant age groups showed that the 95% CI overlap between the groups which implies no statistically significant difference in these two age groups. Table 2 Evaluated parameters for CD4/CD8 ratio for the three infant age groups using DNA PCR as reference standard 0–18 months infant age group (n = 137) 0–11 months infant age group (n = 95) 12–18 months infant age group (n = 42) %Sensitivity 98.7 (CI: 96.1–100) 98.2 (CI: 94.7–100) 100 (CI: 100–100) %Specificity 98.4 (CI: 95.2–100) 97.5 (CI: 92.7–100) 100 (CI: 100–100) %PPV 96.4 94.6 100 %NPV 99.4 99.2 100 %TE 98.5 (CI: 96.5–100) 97.9 (CI: 95.0–100) 100 (CI: 100–100) Abbreviations: n, number tested; NPV, negative predictive value; PPV, positive predictive value; TE, test efficiency.
DNA PCR versus CD4/CD8 ratio
Seventy-five infants of the 76 PCR positive group (98.7%) had CD4/CD8 ratio <1 while 60 infants of the 61 who were PCR negative had CD4/CD8 ratio ≥ 1. All the 9 infants infected in utero had a CD4:CD8 ratio less than 1. Their median CD4/CD8 ratio was similar to that of the rest of the PCR positive babies (0.4; IQR: 0.3–0.6), albeit the numbers are too small for statistical significance considerations. The infant (aged 11 months at specimen collection) who was DNA PCR positive with a CD4/CD8 ratio of 1.1 was from the PACD study. The PCR negative infant (aged 4 months at specimen collection) with a CD4/CD8 ratio of 0.3 was also from the PACD cohort.The overall sensitivity and specificity of the CD4/CD8 ratio were 98.7% (95% CI: 96.1–100 and 98.3% (95% Confidence Interval (CI): 95.1–100) respectively with PPV and NPV of 96.3% and 99.4% respectively and a test efficiency of 98.5% (95% CI: 96.5–100) (Table  2 ). Comparison of all the evaluated parameters between the 0–11 and 12–18 months infant age groups showed that the 95% CI overlap between the groups which implies no statistically significant difference in these two age groups.Evaluated parameters for CD4/CD8 ratio for the three infant age groups using DNA PCR as reference standard0–18 months infant age group (n = 137)0–11 months infant age group (n = 95)12–18 months infant age group (n = 42)Abbreviations: n, number tested; NPV, negative predictive value; PPV, positive predictive value; TE, test efficiency.Discussion In resource-poor countries, the major constraint in the use of DNA PCR for diagnosis of HIV-1 infection in infants under the age of 18 months is the cost of the equipment and the reagents. In addition, highly trained laboratory personnel and stringent quality assurance measures are needed to run this assay for routine diagnosis of HIV infection in infants. In Zimbabwe, enzyme linked immunosorbent assay (ELISA) is routinely performed in both public and private laboratories for diagnosis of various infections including HIV-1. However, because of the transplacental transfer of maternal IgG antibodies, which may persist in infants for up to 18 months, ELISA is not suitable for diagnosis of HIV-1 infection in these infants. Therefore alternative methods are needed for this purpose. In addition, both public and private laboratories have flow cytometers or FACSCount machines for enumeration of T cell subset profile and automated haematological analysers for routine full blood counts with differential. With this equipment available in the country we evaluated the CD4/CD8 ratio as an alternative diagnostic test for infant HIV-1 infection. In our investigation we used Epics XL Coulter flow cytometer equipped with System II software (Beckman Coulter, Miami Florida, USA) for the following reasons. With this instrument, CD4/CD8 ratios can be conveniently obtained. In addition, when used in double platform setting tandem with a haematological analyzer, the results also show absolute CD4 +  cell counts and both %CD4 and %CD8 values among lymphocytes. The %CD4 value among lymphocytes is the recommended parameter for analyzing pediatric samples, as absolute counts for infants are age sensitive and variable. A simpler single platform system such as the FACScount (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA), is not fully suited for pediatric work as it provides CD4 + , CD8 + , CD3 +  T lymphocyte counts and CD4/CD8 ratio but %CD4 are not available. Of note, %CD4 expressed among CD3 +  T lymphocytes, is a different parameter from the customary %CD4 expressed among lymphocytes. The %CD4 expressed among lymphocytes and not the %CD4 expressed among CD3 +  T lymphocytes is recommended for decision making to initiate ARV therapy in children under the age of 18 months [ 5 ]. In Zimbabwe, CD4 +  cell count, is less expensive than PCR and may provide additional information of value to the clinician with respect to prognosis, and the need for prophylaxis and treatment. Optimal flow cytometry for determination of T cell subset profile, offers the added advantage that CD4/CD8 ratio will determine the infection status of the infant while the % CD4 will provide information on decision-making for commencement of HAART. Overall, the CD4/CD8 ratio had a ≥ 98% sensitivity for diagnosis of infant HIV-1 infection and a specificity ≥ 98%. Both sensitivity and specificity were 100% for infants in the 12–18 months age group. Interestingly, in parallel studies performed in 250 HIV-1 seropositive adults, 249 had a CD4/CD8 ratio of <1. The CD4/CD8 ratio of the one patient was 1 at enrollment and has remained so for over one year. When interpreting our data, it is important to note that normal T cell subset values among African children differ from those of other populations [ 14 - 16 ]. A study in Guinea Bissau [ 16 ], reported that Guinean children under the age of 2 years had lower %CD4 and CD4/CD8 ratios and higher %CD8 when compared to their counterparts from developed countries. Interestingly, girls had higher CD4/CD8 ratios and lower %CD8 than boys. In our study, there were no statistically significant differences in absolute T cells, or percentages or CD4/CD8 ratio between boys and girls (Table 3 "refer to  Additional file 1 "). A very few studies in Africa have compared T cell subset profiles between HIV-1 infected and uninfected infants under the age of 2 years [ 14 , 17 ]. Moodley and colleagues in South Africa reported that the most marked changes in lymphocyte subset of HIV-1 infected children aged between 3 and 15 months were a lower %CD4 and higher %CD8 relative to uninfected infants [ 17 ]. In addition, CD4/CD8 ratio was a good predictor of poor clinical outcome at 3 months. The authors concluded that CD4/CD8 ratio and %CD4 among lymphocytes are reliable markers of HIV-1 infection in an African pediatric population. Furthermore a raised CD8 +  cell count rather than a CD4 +  cell count was a more specific prognostic marker of disease progression in HIV infected children. Embree and colleagues, in Kenya, also reported that HIV-1 infected children had lower %CD4 and higher %CD8 by 3 months when compared to uninfected children [ 13 ]. The authors concluded that %CD4 and %CD8 among lymphocytes could be useful as an adjunct in HIV-1 diagnosis. The two African studies mentioned above, have documented the clinical value of %CD4, CD4/CD8 ratio and CD8 counts in HIV-1 infection in infants. In summary, the CD4/CD8 ratio may be useful in identifying infected infants while %CD4 will identify infants who may benefit from cotrimoxazole prophylaxis and/or initiation of HAART, and for management of HIV-infected infants in developing countries in general. We thus propose use of flow cytometry, where available, as a point of care diagnostic tool for ill infants admitted to hospitals with clinical symptoms suggestive of HIV infection and/or AIDS.
Discussion
In resource-poor countries, the major constraint in the use of DNA PCR for diagnosis of HIV-1 infection in infants under the age of 18 months is the cost of the equipment and the reagents. In addition, highly trained laboratory personnel and stringent quality assurance measures are needed to run this assay for routine diagnosis of HIV infection in infants.In Zimbabwe, enzyme linked immunosorbent assay (ELISA) is routinely performed in both public and private laboratories for diagnosis of various infections including HIV-1. However, because of the transplacental transfer of maternal IgG antibodies, which may persist in infants for up to 18 months, ELISA is not suitable for diagnosis of HIV-1 infection in these infants. Therefore alternative methods are needed for this purpose. In addition, both public and private laboratories have flow cytometers or FACSCount machines for enumeration of T cell subset profile and automated haematological analysers for routine full blood counts with differential. With this equipment available in the country we evaluated the CD4/CD8 ratio as an alternative diagnostic test for infant HIV-1 infection.In our investigation we used Epics XL Coulter flow cytometer equipped with System II software (Beckman Coulter, Miami Florida, USA) for the following reasons. With this instrument, CD4/CD8 ratios can be conveniently obtained. In addition, when used in double platform setting tandem with a haematological analyzer, the results also show absolute CD4 +  cell counts and both %CD4 and %CD8 values among lymphocytes. The %CD4 value among lymphocytes is the recommended parameter for analyzing pediatric samples, as absolute counts for infants are age sensitive and variable. A simpler single platform system such as the FACScount (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA), is not fully suited for pediatric work as it provides CD4 + , CD8 + , CD3 +  T lymphocyte counts and CD4/CD8 ratio but %CD4 are not available. Of note, %CD4 expressed among CD3 +  T lymphocytes, is a different parameter from the customary %CD4 expressed among lymphocytes. The %CD4 expressed among lymphocytes and not the %CD4 expressed among CD3 +  T lymphocytes is recommended for decision making to initiate ARV therapy in children under the age of 18 months [ 5 ].In Zimbabwe, CD4 +  cell count, is less expensive than PCR and may provide additional information of value to the clinician with respect to prognosis, and the need for prophylaxis and treatment. Optimal flow cytometry for determination of T cell subset profile, offers the added advantage that CD4/CD8 ratio will determine the infection status of the infant while the % CD4 will provide information on decision-making for commencement of HAART.Overall, the CD4/CD8 ratio had a ≥ 98% sensitivity for diagnosis of infant HIV-1 infection and a specificity ≥ 98%. Both sensitivity and specificity were 100% for infants in the 12–18 months age group. Interestingly, in parallel studies performed in 250 HIV-1 seropositive adults, 249 had a CD4/CD8 ratio of <1. The CD4/CD8 ratio of the one patient was 1 at enrollment and has remained so for over one year.When interpreting our data, it is important to note that normal T cell subset values among African children differ from those of other populations [ 14 - 16 ]. A study in Guinea Bissau [ 16 ], reported that Guinean children under the age of 2 years had lower %CD4 and CD4/CD8 ratios and higher %CD8 when compared to their counterparts from developed countries. Interestingly, girls had higher CD4/CD8 ratios and lower %CD8 than boys. In our study, there were no statistically significant differences in absolute T cells, or percentages or CD4/CD8 ratio between boys and girls (Table 3 "refer to  Additional file 1 ").A very few studies in Africa have compared T cell subset profiles between HIV-1 infected and uninfected infants under the age of 2 years [ 14 , 17 ]. Moodley and colleagues in South Africa reported that the most marked changes in lymphocyte subset of HIV-1 infected children aged between 3 and 15 months were a lower %CD4 and higher %CD8 relative to uninfected infants [ 17 ]. In addition, CD4/CD8 ratio was a good predictor of poor clinical outcome at 3 months. The authors concluded that CD4/CD8 ratio and %CD4 among lymphocytes are reliable markers of HIV-1 infection in an African pediatric population. Furthermore a raised CD8 +  cell count rather than a CD4 +  cell count was a more specific prognostic marker of disease progression in HIV infected children.Embree and colleagues, in Kenya, also reported that HIV-1 infected children had lower %CD4 and higher %CD8 by 3 months when compared to uninfected children [ 13 ]. The authors concluded that %CD4 and %CD8 among lymphocytes could be useful as an adjunct in HIV-1 diagnosis.The two African studies mentioned above, have documented the clinical value of %CD4, CD4/CD8 ratio and CD8 counts in HIV-1 infection in infants.In summary, the CD4/CD8 ratio may be useful in identifying infected infants while %CD4 will identify infants who may benefit from cotrimoxazole prophylaxis and/or initiation of HAART, and for management of HIV-infected infants in developing countries in general. We thus propose use of flow cytometry, where available, as a point of care diagnostic tool for ill infants admitted to hospitals with clinical symptoms suggestive of HIV infection and/or AIDS.Competing interest The author(s) declare that they have no competing interests.
Competing interest
The author(s) declare that they have no competing interests.Authors' contributions LSZ designed the study, analyzed the data and drafted the manuscript. DAK, KJN, AB and GJ participated in the design of the study and preparation of the manuscript. SR conducted the statistical analysis and participated in the preparation of the manuscript. OT performed DNA PCR and analysed the data. AB and CG conducted flow cytometry and analysed the data, with the guidance of GJ. MN and PM participated in the collection of infant specimens and demographic data as well as preparation of the manuscript. All authors read and approved the final manuscript.
Authors' contributions
LSZ designed the study, analyzed the data and drafted the manuscript. DAK, KJN, AB and GJ participated in the design of the study and preparation of the manuscript. SR conducted the statistical analysis and participated in the preparation of the manuscript. OT performed DNA PCR and analysed the data. AB and CG conducted flow cytometry and analysed the data, with the guidance of GJ. MN and PM participated in the collection of infant specimens and demographic data as well as preparation of the manuscript. All authors read and approved the final manuscript.Supplementary Material Additional File 1 Table 3. Gender-based comparison of T cell subset profiles of HIV infected and uninfected babies. Click here for file
Supplementary Material
Additional File 1
Table 3. Gender-based comparison of T cell subset profiles of HIV infected and uninfected babies.Click here for file