Back to Karachi
In 2011 I was able to take a short sabbatical leave and I decided to go to Karachi, my home town. I had been working on an arrangement so I can establish a vaccine Center in Karachi where Pakistan can manufacture its own childhood vaccines and other medicine that can save poor folks, particularly death from snake bites. In 2010, there was a terrible flood in Pakistan that flooded the 1/5th of the whole nation. During this period millions became homeless and migrated en mass. This also brought out venomous snakes out in the open. Both human and the venomous reptiles were distressed and snakes go where to go came in close contacts with humans and caused the loss of thousands of human lives. The absence of anti-snake sera (ASV) and lack of infrastructure to urgently deliver the ASV made the task almost impossible. Therefore, I decided that during my sabbatical, I will tackle two problems: first and foremost to set up an ASV facility and second to sow the seeds for production of the childhood vaccines.
Snakebites and Anti-venom Sera: Generally, reporting of snakebites is not mandatory in many regions of the world therefore, snakebites often go unreported. Consequently, the exact incidences of snake bites are unknown. However, some estimates put the number at 5.4 million snakebites, 2.5 million envenoming, resulting in perhaps 125,000 deaths annually. Others estimate 1.2 to 5.5 million snakebites, 421,000 to 1.8 million envenoming, and 20,000 to 94,000 deaths. Many people who survive bites nevertheless suffer from permanent tissue damage caused by necrosis, neurotoxicity and other enzymatic proteins found in venoms, leading to long-term disability.
Childhood Vaccines Development: Children are especially prone to infectious diseases and many children die from these diseases before reaching the age of 5 years. According to the World Health Organization data, the infectious diseases killed 15 million children out of a total 57 million deaths in 2002. Childhood vaccine is the most cost effective way to prevent illnesses. Vaccination protects not only individuals but sometimes whole communities also from diseases spread by person to person contact. Due to poor sanitation and lack of the supply of clean (safe) drinking water to a large segment of the population , the infant mortality is 7.8 %, very high even in developing countries and life expectancy in Pakistan is only about 60 years (compared to 80 years in the developed counties).
After provision of clean drinking water to all, immunization is the most cost effective health care intervention in a country but it also appears to be largely ignored in Pakistan. It is estimated that about 5 million vaccine doses of (BCG), 12 million doses of DPT, 16 million doses of polio are provided each year to the population. The birth rate is about 3% in the country (4.8 million births each year); therefore, provided vaccine doses are not sufficient even for newly born babies not considering the infant, children and adult population of the country. A reason for less immunization in the country is the cost of most of the vaccines which are mostly imported and are, thus, expensive. Glexo Smith Kline Pakistan Ltd. offers vaccines against hepatitis B and several other infectious diseases including typhoid, hepatitis A and chickenpox. Sanofi-Aventis Pakistan Ltd. provides vaccines against hepatitis A, hepatitis B, measles, mumps, polio, rabies, tuberculosis, typhoid and some other diseases. Amsons Vaccine and Pharma (Pvt) Ltd. is producing vaccine in Pakistan but it is mostly repacking.
Our current emphasis is initially on the production of basic antigens for childhood vaccine: i.e., BCG, DPT, OPV, Measles, Homophiles influenza b (Hib), typhoid fever and pneumococcal conjugated vaccines (PCV-7). All these could be produced relatively easily. The major hurdles are the facilities that can support large scale production of these kinds of vaccines. The other issue is training of personnel who are aware of regulatory issues and quality control. Quality control is a pivotal issue since the products are going into humans.
I had written to one of my colleagues, Dr. Rafiq Khanani regarding these ideas. He was the head of Pathology at Dow University of Health Sciences (DUHS) and the president of infection control in Pakistan, Sindh province. He agreed to offer me a place and support to carry out this initial work. The Vice Chancellor of the University at that time, Dr. Masood Hamid Khan turned out to be an excellent leader and a man of vision. Under his leadership DUHS has flourished and has developed numerous centers. Therefore, a Center for Vaccine Development would be a Jewel of the Crown for him.
Development of Anti-Snake Venom Sera (ASV) in Horses
The development of anti-snake venom immunotherapy (ASV) has a degree of urgency in Pakistan. According to the World Health Organization (WHO) and Ian Simpson’s reports, who came to assist Pakistan a decade or so years ago, Pakistan has the second highest incidence of venomous snakebites, ranging from 20,000-30,000/year (second only to India). Generally, venomous snakebite-mortality ranges from 10-15%, depending on the degree of remoteness of the area where an incident occurs, type of snakebite, site of bite and availability of proper primary care and ASV within a very narrow window of time. In my opinion, after speaking with snake handlers, travelling for years in the Sind province, and travelling as a monk when I was 16 years of age, I believe that the incident reports are a gross underestimate. In Pakistan, there is no real reporting system of anything (including mortality, cancer, heart attack or strokes) and generally a snake-bitten child or adult does not go to a doctor in the remote rural area but seeks help from a monk, a faqir, a mulana or a Veda who sometimes do have remedies (mostly worthless, though). Otherwise, it is left up to God’s will to save the poor bitten souls. I state this in order to bring home the point that if DUHS develops ASV it needs to think about how many doses are needed and to be produced how to distribute them to the remotest and the rural parts of Pakistan, and how to create an awareness of the availability of the AVS. DUHS needs to produce at least 10 times more vials of ASV than what people of Pakistan need annually in order to reduce the mortality at significant levels. This means a minimum of 300,000 doses of ASV will be needed annually. Higher numbers are needed for the widespread distribution as well as need for multiple doses in many cases. The need for wide distribution in every little town is necessary since the cobra bite can kill a person in 15 minutes or so (as we unfortunately witnessed in one of the horses (Devdas) we immunized and gave him a slightly higher dose than what we estimated from the rabbit and rodent injections, but acting in accord with the WHO recommendation).
Figure1: Showing our first Immunizations on four horses with VC Prof Dr. Masood Hameed Khan, Dr. Omar Bagasra, Dr. Rafiq Khanani and Dr. Zameer Ahmed and others.
The cost of each vial of 1.00 mL of ASV would be around 100 Rupees/vial (I estimate that the initial cost would be little higher but after that it would be less than 50 Rupees/dose. In the US dollar it will be close to 50c). The current retail cost of the Indian-made ASV is between 1,800 to 2,200 Rupees. I think we can easily prepare around 250-300, 000 doses of the ASV in 15-20 horses and keep the retail price around 700-800 Rupees/vial. This will cover almost all the areas of Sind Providence. The estimated profit would be between >18 Karor Rupees/year (or $18M US).
- a) Technical Issues Related to Immunization and Hyperimmunization: The initial immunization and hyperimmunization requires a highly scientific protocol. In addition, it is better to develop multi-valent vaccine than a mono-or divalent vaccine. In this case one would need an excellent immunologist who knows the field well enough. It cannot be left up to a veterinarian doctor to do this task. Firstly, the immunization MUST be carried out with low, safe and diluted doses of natural proteins and toxins. Heat inactivation, UV or other treatments would denature the poisons, as well as make them inactive; the sera obtained from these animals would be essentially useless, although it may provide excellent immunoprecipitation. This is a simple rule of immunology that was worked out over a hundred years ago by Landsteiner, (who also discovered blood group). Similarly, the IgG should not be digested or manipulated since it will significantly decrease the efficacy of the ASV.
In case of Cobra, we need to make minor adjustment and may need to attenuate the toxin a bit to be safe for the horses. A short bust of 56o C treatment may render the most neurotoxic part of the venom inactive. We already have tested this in rabbits and will carry out the immunization in a safe manner.
- b) Immunization Schedules: The immunization should be carried out with appropriately diluted snake venom with an adjuvant, initially and preferably with the incomplete Freud Adjuvant. The secondary immunizations or the booster doses should be scheduled 4-6 weeks after the primary immunization with the incomplete Freund Adjuvant or oil. After that, if animals are healthy, they should be immunized every 3 weeks. The blood should be drawn at 6 weeks after the primary immunization, and 2 weeks after every immunization, with one week of rest and recuperation for the horse before the subsequent immunization. Generally, after the primary immunizations, the animals should be immune to the snake venoms and theoretically should have no adverse effects.
It should be left up to the judgment of the veterinarian doctor to look after the horses and have him/her decide how much plasma would be safe to obtain from each horse. Ten percent volume is generally safe but if the horse is older, small, and weak from previous plasmaphoresis, it should be given sufficient time to recover from the plasma isolation. Hypovolumic shock should always be avoided at all costs.
- c) Periodic Analyses of the immune ASV: It will be imperative to test plasma from each horse for ASV using both immunoprecipitation as well as the ELISA method. I trained Dr. Zameer to carry out these very important assays, and to keep a strict record of each horse and the type of snakes’ venom each was immunized with. A simple error in this procedure can result in serious and adverse outcomes at the clinical end. The ASV titration by both methods would be necessary to develop a high quality ASV. Most importantly, ASV must be tested for efficacy by injecting mice with diluted but lethal doses and ASV subsequently.
- d) Development of multivalent ASV: There are two options in the development of multivalent sera that would be useful. I suggested that we only immunize separate horses for each of the snake venoms, and not the multiple venoms. After each of the horses has developed ASV for specific snake venom, we have the option to pool two or more sera to develop multivalent sera. The second option is to immunize each horse with each of the venoms one after another. I believe this is a bad option and may harm the horse being immunized adversely.
- e) Collection of Snake Venoms: We constructed a sepentarium. However, our best source of snake venoms was the snake catchers who bring a large number of venomous snakes from the wild and milk the venoms for us on a regular basis.
Figure 2: Milking Cobra and Viper
Figure 3: Learning how to extract venom from Viper and Cobra.
Childhood Vaccine Center: My emphasis was emphasis to initially produce basic antigens for childhood vaccine: i.e., BCG, DPT, OPV, measles, homophiles influenza b (Hib), typhoid fever, and pneumococcal conjugated vaccines (PCV-7). All these could be produced relatively easily. The major hurdles are the facilities that can support large scale production of these kinds of vaccines. The other issue was training of personnel who are aware of regulatory issues and quality control. Quality control is a pivotal issue since the products are going into children. In order to acquire proper equipment and supplies I had travelled to Lahore to determine the suitability of a Bioreactor as well as other equipment.
Current Status: We acquired a Bioreactor and minimum space and facilities to initiate the production of BCG vaccine. We decided on BCG (Bacillus of Calmette and Guérin) because we believe that it can be achieved with relative ease as well as a high quality; also, BCG can be produced in a short time.
We also set up a tissue culture facility, and a BSL level-3 anaerobic room and a BSL level-2 aerobic room to facilitate future vaccine production.
Figures 4: BioReactor for BCG and Training of Cell-Culture Methods for Students
DUHS Graduate Program and Facilities
Early after my arrival at DUHS, I started to look at its graduate programs. I looked at the Curriculum Vitas of the Pathology Faculty, and assessed their capacity and caliber. I was amazed to see that out of a large number of faculty, only very few were theoretically qualified to teach graduate school (please see my confidential remarks in Appendix I). Most of the teachers are undergraduates themselves (MBBS), and have M. Phil degrees (equivalent of a Master in Science in the US or UK), which hardly makes them qualified to teach doctoral level courses and students. In the USA, we have an accreditation body that evaluates such matters and it would be a good idea if the Government of Pakistan were to develop such a national body of evaluators who can, without bias, determine the credibility of the teaching, degree-granting colleges and universities (suggested name would be “Pakistani Association of Colleges and Universities” or PACU). During our first meeting on 23 February 2011, and during our subsequent meeting on 7 March, I brought forward some of the major concerns, and shared my view on numerous issues including how to redesign the graduate school. These included the following suggestions:
I explained at that time that DUHS badly needs an overhaul of the graduate program, and needs to hire two new tenure-track faculty, and reformat the courses and faculty for this purpose. At that time, you approved advertisement for the proposed positions as well as some sort of attendance policy for the current faculty who arrive late and leave by 2 PM. There appears to be no real drive among the faculty to train the students. Lack of faculty motivation has promoted the same among students. Since my earlier observation, I am even more concerned regarding the irresponsible behavior of DUHS faculty. Generally, I have observed that 50% of the classes are cancelled without any reason. Students come to class and wait for 30-40 minutes only to learn that class has been cancelled. More disconcerting yet is the news that scheduled exams are delayed for days or even weeks. One of our students who related this to me has a young daughter who arranged for a babysitter until 1 PM. The exam was scheduled for 10 AM, but the examiner did not arrive until 12 PM. Then, the students were told that the exam was not ready, and that they should return at 2 PM. When they arrived at 2 PM, they waited and then after one hour were told that the exam would be tomorrow. One of the students has taken exam more than a year ago and his grades have not been posted yet!
I suggest that we implement a strict time table for the M.Phil as well the Ph.D. program. Please see the Appendix II for details.
The medical education program is also not up to professional standards. I was invited to give a few lectures on forensic science to the third-year medical students by Dr. Mukkaram Ali. The class was supposedly to start at 8:30 AM. I was anxious to start and arrived at 8:25 AM. At that time, essentially no one had arrived—staff, faculty, or attendants. The elevator was even shut down. The lecture room was locked, and the attendant who had the key arrived at 8:40 AM. Then the students began to arrive. Dr. M. Ali arrived at 8:51 AM, and asked me to wait for the students who, he said, generally arrive at 9 AM. I started to talk to some of the students who were there before 9 AM since I think a teacher should not wait for the students to come; if anything, it should be the other way around. It was only a 50-Minute class and some of the students arrived at 9:28 AM. Less than 50% of the students attended the class. After class, several students stayed and chatted with me with various questions. So, it was not that students were uninterested. I think it is the culture of teaching by the faculty that is faulty. I gave two lectures on forensics, and the second time (when students knew I was going to teach them) they were in the lecture room before I got there!
In addition, DUHS needs an USML preparatory software for DUHS for all the DIMC students.
This is difficult task, and to change the current culture would require firm and consistent implementation of rules and regulations. It would be worthwhile to let the teacher know that they are working for an organization and getting paid for it.
- Teaching Activities
Since my arrival, I have been teaching graduate students at various levels. I started to teach them a full course on immunology, and have delivered weekly courses, sometimes three-hours long, on various aspects of immunology. Initially, it was designed to teach Ph.D. candidates only but word spread about the course, and now I have most of the M.Phil students as well. Sometimes, students from other universities attend my classes. My final three-hour lecture is scheduled for Wednesday, 13 April.
I have given numerous lectures to graduate students on such subjects as tissue regeneration, experimental design, ethics, gene alignment tools, real time PCR (polymerase chain reaction), just to mention a few.
I also noticed that DUHS has beautiful buildings but no real research lab. I recommended the creation of a fully functional research lab. I have recommended the purchase of a gene box, as well as tissue culture, and a PCR system. It is my understanding that the funds for this task are being donated by the former Dow graduates residing in the USA (please see Appendix III for the list of needed equipment). Research is the only engine that drives the higher learning system and it should get the top priority.
In order to acquire proper equipment and supplies I had travelled to Lahore to determine the suitability of a Bioreactor as well as other equipment.
After four years, the DUHS centers are producing ASV but not license to distribute it yet and has successfully produced BCG at large scale.