Evaluation of cold chain monitoring in Kelantan, Malaysia.

Evaluation of cold chain monitoring in Kelantan, Malaysia K. Hanjeet,1 M.S. Lye,2 M. Sinniah,3 & A. Schnur4 An analysis was carried out on a total of 883 cold chain monitor (CCM) cards, which had been attached to batches of poliomyelitis, measles, DPT (diphtheria, pertussis, tetanus) and hepatitis B vaccines, during their transport and storage from the central store in Kuala Lumpur to Kelantan, a state in north-eastern Malaysia; 234 freeze watches attached to hepatitis B vaccines were also analysed. The monitor cards and freeze watches were observed at six levels between the central store and the periphery during distribution of the vaccines, and a colour change in any of the four windows (A, B, C, D) on the CCM cards or the freeze watches was recorded. In addition, 33 unopened vials of oral poliovirus vaccine (OPV), collected from refrigerators in 29 health facilities in Kelantan, were tested forpotency using the tissue culture infective dose 50 (TCID50) method; 14 of them (42%) did not meet the WHO criteria for potent vaccines. The results showed that at the final destination 13.4% of all cards remained white while a colour change to blue was observed in 65% in window A, 16.6% in window B, and 4.4% in window C; none had turned blue in window D indicating that the vaccine had not been subjected to temperatures -340C for 2 hours. All but 2 of the 234 freeze watches had turned purple, which indicates exposure of the hepatitis B vaccines to temperatures below 0°C. These results will assist health planners to correct the weaknesses identified in the cold chain system. Introduction Immunization is a powerful and very cost-effective weapon of modern medicine for the prevention of major childhood diseases. In Malaysia, immuniza- tion is routinely given against such diseases of child- hood as tuberculosis, diphtheria, tetanus, pertussis, poliomyelitis, measles, rubella and hepatitis B. In 1992 the infant (under 1 year) mortality rate and toddler (1-4 years) mortality rate in Peninsular Malaysia were 11.6 and 0.8 per 1000 live births, re- spectively (1). Immunization coverage data for 1992 are shown in Table 1. Among the Health for All goals by the year 2000, as described in WHO's Ninth General Pro- gramme of Work (1996-2001), are maintenance of a high level of immunization coverage (at least 90% of children under 1 year old), reduction of measles cases (by 90%) and deaths (by 95%), elimination of neonatal tetanus (i.e., no longer of public health I Public Health Specialist, Department of Community Medicine, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia. Requests for reprints should be sent to this author. 2 Consultant Epidemiologist, Department of Community Medicine, Institute for Medical Research, Kuala Lumpur, Malaysia. 3 Consultant Medical Microbiologist, Department of Tropical Medi- cine, Institute for Medical Research, Kuala Lumpur, Malaysia. 4 Technical Officer, United Nations Office, Manila, Philippines. Reprint No. 5716 significance in terms of incidence and severity), and global eradication of poliomyelitis (2). Although routine immunization has greatly reduced the incidence of these diseases, outbreaks have oc- curred from time to time. This raises questions on the effectiveness of the immunization programme, for the proper functioning of which the cold chain is vital. High coverage would have little meaning if the vaccines administered had lost their potency (3). To the best of our knowledge this is the first study in south-east Asia using cold chain monitors to evaluate the effectiveness of the cold chain. It pro- vides useful information to health planners and policy-makers for strengthening the cold chain in the national immunization programme. Table 1: Immunization coverage in Malaysia in 1992 Vaccine type Coverage (%) BCG 98.4 OPV-I 91.2 OPV-Ill 90.4 Measles 79.6 DPT-I 91.7 DPT-I1 91.3 HBV-I 94.8 HBV-11 90.4 HBV-III 86.7 Bulletin of the World Health Organization, 1996, 74 (4): 391-397 © World Health Organization 1996 391 K. Hanjeet et al. Site and methods Kelantan, one of Malaysia's 13 states, is situated on the north-east coast of the peninsula and has a dry and a wet season, with little variation in temperature throughout the year (range: 22-34°C). All ten dis- tricts in the state were included in this study; all 248 health facilities are linked by road except for a few that can only be reached by river. Vaccines from the central national store in Kuala Lumpur are always sent by air to the state-level store, but local distribu- tion within the state is mostly by 4-wheel-drive motor vehicles. The vaccines are packed with ice and sawdust for transport and during distribution. In this study, no changes or adjustments were made in the routine vaccine supplies and flow, transportation, and usage. The staff involved in management of the cold chain had been trained in cold chain monitoring in April 1990 during a national workshop, as well as on the site by a WHO team. Methodology Cold chain monitor (CCM) cards (3M type) and freeze watches (FWs) (Coldmark type) are used to monitor the vaccines. Each CCM, which is effective in indicating whether the vaccines were exposed to higher than recommended temperatures, has a heat- sensitive indicator in the form of a strip with four windows (A, B, C and D in Fig. 1). The indicator operates above two different temperatures (10°C and 34°C). The higher the temperature above the CCM threshold, the more rapidly the colour changes to blue, which is irreversible even when exposed to lower temperatures again. The manufacturer recom- mends that the CCM card be refrigerated for at least 30 minutes before activation. Activation is carried out by pulling out the tab on the left-hand side of the strip. Colour changes were recorded at various tran- sit levels along the vaccine route from level 1 (central national store) to level 6 (peripheral health facility). Levels 1 and 2 are the national store and state store, respectively, for vaccine storage purposes only. Vac- cine usage for immunization starts at level 3, with maximum usage at levels 4 and 5. On the back of each CCM card are given instructions to interpret the readings (Fig. 1). The Coldmark freeze watch is used to monitor the temperature of tetanus toxoid, triple antigen and hepatitis B vaccines, which should not be exposed to Fig. 1. Cold chain monitor (CCM) card: (left) front and (right) back views. WHO Bulletin OMS. Vol 74 1996392 Cold chain monitoring in Malaysia temperatures below 0°C. It consists of a bulb at the end of a glass tube, which remains colourless at tem- peratures above 0°C but changes to purple at and below 0°C. Vaccines are routinely dispatched by manufacturers to the central national store in Kuala Lumpur with a CCM card and FW (one CCM and/or one FW per 3000 doses). However, for the purpose of this study additional CCMs and FWs were added at the central store for vaccines that were destined for Kelantan (Fig. 2). In Malaysia the frequency of dispatch to periph- eral levels varies, being once in 3 months, once a month, fortnightly or weekly depending on the de- mand, the available storage facilities, and the dis- tance from the health centre. Vaccines are also stored over similar periods at the periphery where, as a matter of policy, an additional 25% of the orders is held in reserve at all times. The CCMs and FWs were attached to the various types of vaccines in their original packing, without excessive handling of the vaccines; 1500 CCMs were attached to poliovirus, measles, DPT (diphtheria-pertussis-tetanus) and hepatitis B Fig. 2. Flow-chart for cold chain monitors. Manufacturer Central store, Kuala Lumpur (1500 cold chain monitors attached) (sent quarterly) State-level store, Kelantan (sent monthly) District hospitals (sent monthly) Main health centres Health subcentres Health subcentres Health subcentres (sent weekly) (sent weekly) + c i Community clinics Midwife clinics WHO 96249 Community clinics Midwife clinics (as and when there is a delivery)i Homes vaccines (HBV), the last named carrying also 300 freeze watches. These were distributed following the routine schedule to the state store in Kelantan. On receipt at the various transit levels, the monitor cards were read and recorded. Once the vaccines had been completely used up, the completed cards were collected and stored at room temperature until analysis. To test the effectiveness of the cold chain sys- tem, 33 samples of oral poliovirus vaccine (OPV) and 3 of freeze-dried measles vaccine from the peri- pheral transit levels were carefully packed in ice and transported in insulated flasks to the Institute for Medical Research (IMR) for potency testing using Hep-2 cells (Cincinnati line, passage number 191). Each of the poliovirus types in the OPV was titrated separately using heterologous type-specific polio- virus antisera for neutralizing the other viral types present. For each titration, a reference vaccine strain stored at -20°C was included as standard. The poliovirus vaccine titration endpoint was calculated by the Spearman-Karber formula and expressed as TCID501 per dose (4). The origin of these 33 polio vaccine samples was as follows: state store (1), dis- trict hospitals/integrated stores (3), outpatient de- partment (1), main health centres (6), health subcentres (13), maternal and child health centres (4), and community clinics (5). Measles vaccine titration was performed using Vero cells and standard freeze-dried reference vac- cine; the endpoint was calculated by the Spearman- Karber formula (4) and expressed as log10 TCID per dose. Results The data were analysed using WHO-EPI software. Cold chain monitor. Of the total of 1500 CCM cards distributed, 883 (59%) were available for analysis by colour index on arrival at the various transit and final destinations (Table 2). At level 1, for example, 0.3% had turned blue in window A; no monitors were blue in windows B and C. At level 6, however, 65.6% of the CCMs had turned blue in window A, 16.6% in window B (from 0% at level 1), and 4.4% in window C (from 0% at level 1). Table 3 shows the propor- tions ofCCM cards that turned blue during transpor- tation and storage at each level; the percentages are not cumulative. The levels with the highest propor- tion of cards turning blue in window B during stor- age were at level 3 and level 4 (5.7% and 5.1%, resp.) (Table 3). Colour changes in windows A and B oc- curred more frequently during storage than during transportation. WHO Bulletin OMS. Vol 74 1996 393 K. Hanjeet et al. Table 2: Percentage of CCM cards with colour change in windows A to D on arrival at different levels of vaccine distribution Windows with colour change (%): Place Level White A B C D Central medical store, Kuala Lumpur Li 99.7 0.3 0 0 0 State medical store, Kelantan L2 83.3 10.3 6.3 0.1 0 District hospital L3 62.0 31.1 6.6 0.3 0 Health centre L4 35.5 56.7 7.6 1.2 0 Health subcentre L5 24.3 59.7 14.1 1.9 0 Community clinic L6 13.4 65.6 16.6 4.4 0 Table 3: Percentage of colour change during transport and during storage at various levels Colour change during transport (%): Colour change during storage (%): Place Level Route White A B C Storage White A B C Central medical store, Li Supplier to Li 99.7 0.3 0 0 In Li 96.1 3.1 0.8 0 Kuala Lumpur State medical store, L2 Li to L2 94.8 4.5 0.7 0 In L2 88.1 11.5 0.4 0 Kelantan District hospital L3 L2 to L3 90.7 9.1 0.2 0 In L3 79.0 14.2 5.7 1.1 Health centre L4 L3 to L4 92.8 7.0 0.2 0 In L4 78.2 16.1 5.1 0.5 Health Subcentre L5 L4 to L5 95.7 3.3 1.0 0 In L5 81.7 16.3 1.7 0.3 Community clinic L6 L5 to L6 92.1 7.2 0.7 0 In L6 81.4 16.7 1.9 0 Table 4 shows the percentages of CCM cards were within acceptable limits. Table 4 also shows the subjected to delay during transport of vaccines from duration of storage at each transit level; 56% of the the supplier to level 1 and onwards to level 6. A vaccines were stored for more than 2 weeks in level delay of more than 6 days for 94% of vaccine dis- 1, and nearly 60% for more than a week in level 2. patches was noted between the supplier and level 1. From level 3 to level 6 between 31% and 21% of The transport times beyond level 1 (range: 1-2 days) vaccines were stored for less than a week. Table 4: Percentage of delays (days) during transport between levels, and of delays (weeks) during storage at various levels Days: <1 1 to <2 2 to <3 3 to <4 4 to <5 5 to <6 >6 Route: Supplier to Li 0 0 0 0.2 5.5 0.3 94.0 Li to L2 8.2 42.8 46.6 0.8 0 0.2 1.4 L2 to L3 89.0 5.1 2.2 0 0 0.3 3.4 L3 to L4 95.5 1.1 0 0 1.1 0.3 2.0 L4 to L5 90.7 0.7 0 2.2 0.7 0 5.7 L5 to L6 91.4 0 1 1.9 0 1.9 3.8 Weeks: <1 1 to <2 2 to <3 3 to <4 4 to <5 5 to >6 >6 Storage in: Li 3.1 6.0 56.1 13.6 2.0 1.5 17.3 L2 40.5 13.1 15.5 9.5 3.3 2.1 16.0 L3 31.4 13.9 8.5 7.7 8.5 6.9 23.1 L4 27.3 16.4 9.7 8.2 10.3 2.9 25.2 L5 23.5 15.6 8.6 10.4 7.3 6.4 28.2 L6 21.8 10.9 13.5 7.1 11.5 6.4 28.8 WHO Bulletin OMS. Vol 74 1996394 Cold chain monitoring in Malaysia Table 5: Potency titres of 33 batches of poliovirus vaccines type 1 (P1), type 2 (P2) and type 3 (P3), compared with indications on the CCM cards Log1o TCID per 0.1 ml Expiry date State of CCM No. Batch No. (month/year) Type Pla Type P2a Type P3a Potency CCMb 001 S 1448A4B 3.92 5.4 4.6 5.0 Low No card 002 S 1386A4A 9.91 5.4 4.6 5.1 Low No card 003 S 1431A4A 2.92 5.6 4.6 4.8 Low No card 004 S 1492A4C 12.92 5.6 4.9 5.0 Pass No card 005 S 1492A4B 11.92 5.8 4.9 5.0 Pass A, B 006 S 1492A4B 11.92 5.8 4.7 5.0 Pass A, B 007 S 1448A4B 3.92 5.6 4.9 5.1 Pass A, B 008 S 1448A4B 3.92 5.7 5.1 5.4 Pass No card 009 S 1431A4A 2.92 5.9 4.9 5.4 Pass No card 010 S 1431A4A 2.92 5.6 4.8 5.1 Pass No card 011 S 1448A4B 3.92 6.0 4.8 5.4 Pass No card 012 S 1492A4B 11.92 6.1 4.7 5.2 Pass No card 013 S 1386A4A 9.91 5.2 4.3 4.4 Low A 014 S 1492A4B 11.92 5.9 4.8 4.8 Low A, B 015 S 1492A4C 12.92 5.8 4.7 5.1 Pass No card 016 S 1492A4C 12.92 6.1 4.5 5.1 Pass No card 017 S 1431A4A 2.92 5.6 4.6 4.8 Low No card 018 S 1492A4C 12.92 5.7 4.7 5.1 Pass A 019 S 1492A4C 12.92 5.9 4.7 5.0 Pass A, B 020 S 1492A4C 12.92 5.5 4.5 4.6 Low A 021 S 1448A4B 3.92 5.7 4.5 4.7 Low A 022 S 1492A4C 12.92 5.9 4.7 4.8 Low A 023 S 1492A4B 11.92 6.1 4.5 4.7 Low A, B 024 S 1492A4C 12.92 5.7 4.6 4.6 Low A, B 025 S 1492A4C 1.93 5.1 4.5 5.0 Low A, B, C 026 S 1448A4B 3.92 5.5 4.5 5.0 Pass A, B 027 S 1492A4C 12.92 5.8 4.4 4.9 Low White 028 S 1492A4C 12.92 5.8 4.5 5.3 Pass A 029 S 1492A4C 12.92 5.8 4.6 5.4 Pass White 030 S 1492A4C 12.92 6.0 4.5 5.0 Pass A 031 S 1448A4B 3.92 5.8 4.6 5.4 Pass No card 032 S 1448A4B 3.92 5.9 4.8 5.1 Pass No card 033 S 1492A4B 11.92 5.6 4.1 4.7 Low A, B, C a Minimum titre for a single human dose should not be less than 106, 105 and 105.5 for poliovirus types 1, 2 and 3, respectively. According to the WHO criteria, the assay should not differ by more than 1005; titres below this point are indicated in bold. b A, B and C indicate the windows in the CCM cards that had turned blue. See text for possible explanation of discrepancies. Of the total of 300 freeze watches that accompa- nied the HBV vaccines, 234 were available for analy- sis. Except for two, all (99%) had turned purple at the state-level store. Potency. The potency of 33 vials of poliovirus vac- cine selected from various health centres was tested in the IMR using the TCID50 method. Fourteen vials (42%) showed low potency according to the WHO criteria of potency testing (Table 5), according to which the minimum titre for a single human dose of Sabin trivalent vaccine should not be less than 106 infectious units for type 1 poliovirus, 105 infectious units for type 2 poliovirus, and 1055 infectious units for type 3 poliovirus (5). At the 95% confidence interval the assay should not differ by more than 100-5. Of the 14 vials of polio vaccine showing low potency, only three had tested low on more than one poliovirus type. All three vials of measles vaccine tested had titres above the WHO-recommended values. Mea- sles vaccine is also more heat stable than polio vac- cine because it is distributed in freeze-dried form and is reconstituted just before use. Discussion Some weaknesses were identified through this study. Window A, for example, in 65.6% ofCCM cards had turned to blue by the time the vaccines had reached the peripheral health centre (level 6); another 16.6% had turned blue at window B and 4.4% at window C by the time they reached level 6. This is an indica- tion that the cold chain in Malaysia needs strength- ening. Appropriate storage and transportation of vaccines are of utmost importance for maintaining their potency. Our study showed that the risk of WHO Bulletin OMS. Vol 74 1996 395 K. Hanjeet et al. allowing HBV to freeze was greatest at the state- level store. Inactivated vaccines cannot be identified except by potency tests, which are not widely available in Malaysia. Only large volumes of vaccines, when sus- pected, are sent for potency testing which is very expensive and laborious to carry out; it is usually cheaper to discard small stocks of suspected vaccine. A defective vaccine may only become apparent when a vaccinated person acquires the disease the vaccine was supposed to prevent (6). There is cur- rently no convenient method for monitoring vaccines, except by using cold chain monitor cards and freeze watches. Most sensitive to high tempera- tures are the live viral vaccines (e.g., poliovirus and measles vaccines); killed vaccines and toxoids are generally more tolerant of higher temperatures with no refrigeration but can be destroyed by extreme temperatures, particularly freezing. A higher pro- portion of cards turned blue in window B at transit levels 3 and 4 which represent the district-level stores and the main health centres, respectively. Some dis- trict stores are a long distance away from the state- level store so that transport of vaccines by road can take 6-8 hours in temperatures of 30-32°C. Similar findings were reported in a European country, but in that study the vaccines were distributed by post (7). Vaccines transported from the manufacturer to level 1 remained satisfactory although 94% of them took 6 days or more to arrive. Their storage at level 1 was also satisfactory, with only 3.3% of cards changing to blue at window A and 0.8% of cards changing to blue at window B. In a study in India (8) it was reported that most of the cards turned blue at level 1. Most of the vaccines were stored for more than two weeks in level 1. At level 2 the storage time was much less; about 40% were stored for a week or less, the vaccines being passed on to levels 3 and 4. The current guideline is that there should be 20-25% reserve of vaccines at every level at all times for use in cases of emergency and vaccine delay. In a study in Egypt (9), it was reported that many areas had no vaccines in stock, which means that a number of children must have been turned away because there was no vaccine. This reduces immuni- zation coverage and prevents the realization of our objective to immunize >90% of the susceptible population. Poliovirus vaccine is the most heat sensitive of all the vaccines; the trivalent vaccine contains poliovirus types 1, 2 and 3 which must meet the minimum viral titres recommended by WHO for each viral type. Even if only one viral type falls be- low the recommended titre, the vaccine is considered to be not potent. Using these stringent criteria, 42% of the 33 vials of vaccines did not pass the potency test (Table 5). To explain discrepancies between this result and the colour change in windows A, B and C on the CCM card it is possible that the vaccine and the CCM card had been separated at some point during transit or that an older vial was attached to the CCM card and sent for potency testing. In a study carried out in India in 1992, poliovirus vaccines were tested at various temperatures and were found to be potent despite the change in windows A and B (V.B. Mandke et al., 1991, unpublished).a Although 1500 CCM cards had been sent out, only 883 were received; thus, about 41% were lost or misplaced, or thrown away, perhaps because they had turned blue. This was the first time some people at the peripheral level had seen these cards, which they may not have filled in properly; or they may have been afraid to be interviewed since training to fill these cards had been conducted earlier. A similar situation was not reported in studies carried out in England (6), Hungary (7), and India (8). All the 300 freeze watches which accompanied the hepatitis-B vaccines into the refrigerator in the state-level store showed temperature variations within the refrigerator with a colour change in those placed at the back of the refrigerator. As the maximum-minimum thermometer did not show freezing temperatures, it is difficult to say what went wrong. Subsequently, only a few of the freeze watches followed the hepatitis B vaccine to the peripheral level. This study has brought to light many weak- nesses in the cold chain, such as absence of contin- gency plans during power cuts, or inadequate training of health staff, pharmacists, storekeepers and attendants in cold chain maintenance pro- cedures. Sometimes there were problems when vaccines were not available, or because of old re- frigerators, or when there was no cold room in the state-level store, or inadequate equipment to store a large quantity of vaccines. Some thermometers were not in working order, or vehicles for transporting vaccines were not air-conditioned; some centres did not keep temperature records or had expired vac- cines in stock, and potency tests were not carried out regularly. The results of this study should assist man- agers and health planners in the rectification of these conditions which will definitely improve the cold chain. a Editorial comment. WHO/EPI does not recommend the use of cold chain monitors to provide an indication of the vaccines' po- tency. As their name implies, the CCMs are used to monitor the cold chain and warn of failure at any point. The potency of the vaccine cannot be determined by the status of the cold chain monitor. 396 WHO Bulletin OMS. Vol 74 1996 Cold chain monitoring in Malaysia Acknowledgements We thank the Director-General of Health in Malaysia, Tan Sri Dato Dr Abu Bakar bin Dato Suleiman, for permission to publish this paper, as well as the Kelantan State Direc- tor of Medical and Health Services and his staff, especially Dr Azmi Hashim and Matron Fatimah Dolmat, for their cooperation in this study. We also acknowledge support from the World Health Organization in providing national training in maintenance and surveillance of the cold chain. This project was funded by the Malaysian Ministry of Sci- ence Environment and Technology (grant No. 90-4 IMR). Resume Evaluation de la surveillance de la chaine du froid dans l'Etat de Kelantan, Malaisie La Malaisie ayant un climat chaud et humide toute l'annee, avec des temp6ratures comprises entre 220C et 340C, il a 6t6 proc6d6 en 1991-1992 a une 6valuation du systeme de surveillance de la chaine du froid et au contr6le de I'activit6 des vaccins utilis6s dans le cadre du Programme 6largi de vac- cination. Au total, 883 cartes de contr6le de la chaine du froid (CCM) jointes a 4 types de vaccins (poliomy6lite, rougeole, DTC (dipht6rie, t6tanos, coqueluche) et hepatite B) transportes du d6p6t central de Kuala Lumpur a l'Etat de Kerantan, ont ete analys6es; 234 indicateurs de cong6lation joints au vaccin antihepatite B ont 6galement 6te analyses. Les cartes de contr6le de la chaine du froid ont et6 observ6es a diverses etapes du trans- port, tout changement de couleur dans l'une des fenetres (A, B, C, D) 6tant not6. Tout changement de couleur de l'ampoule de l'indicateur de con- g6lation a egalement 6te not6. L'activit6 de 33 flacons non ouverts de vaccin antipoliomy6litique pris dans les refrig6rateurs de 29 6tablissements de sante de l'Etat a 6t6 deter- min6e a l'institut de recherche medicale de Kuala Lumpur par la methode des DICT50; 14 d'entre eux (42%) ne repondaient pas aux normes OMS d'ac- tivite des vaccins. L'observation des cartes de con- tr6le au point de livraison des vaccins a montre que 13,4% des cartes 6taient rest6es blanches tandis que 65% avaient vire au bleu dans la fenetre A, 16,6% dans la fenetre B, 4,4% dans la fenetre C, et aucune dans la fenetre D. Tous les indicateurs de cong6lation sauf 2 avaient vir6 au violet, ce qui indi- que que les vaccins antihepatite B ont ete exposes a des temp6ratures 6gales ou inf6rieures a 00C. Les points faibles reperes au niveau de la chaine du froid 6taient I'absence de plan de secours en cas de coupure d'6lectricite et l'insuf- fisance de la formation du personnel concernant l'int6grite de la chaine du froid. Les problemes constat6s au niveau du transport et du stockage 6taient dus au mat6rial trop vetuste pour assurer le maintien des temp6ratures optimales, a I'absence de chambre froide dans le d6p6t central de l'Etat, au non-fonctionnement des thermometres, a I'ab- sence d'enregistrement quotidien des temperatures et parfois, dans certains centres, a la pr6sence de vaccins p6rim6s. Ces insuffisances devront etre corrig6es dans les meilleurs d6lais. References 1. Annual report. Kuala Lumpur, Ministry of Health, 1992. 2. World Health Organization. Ninth generalprogramme of work covering the period 1996-2001. Geneva, 1994: 19-20 ("Health for All" Series, No. 1 1). 3. Singh H et al. Extent of occurrence of the six vaccine preventable diseases in vaccinated/unvaccinated chil- dren. Indian pediatrics, 1991, 28: 635-639. 4. Finney DJ. Statistical methods in biological assay. London, Charles Griffin, 1952. 5. Requirements for poliomyelitis vaccine (oral). Adden- dum 1987. In: WHO Expert Committee on Biological Standardization. Thirty-eighth report. Geneva, World Health Organization, 1988 (WHO Technical Report Series, No. 771). Annex 4: 91-92. 6. Casto DT, Brunell PA. Safe handling of vaccines. Pediatrics, 1991, 87: 108-112. 7. Lugosi L, Battersby A. Transport and storage of vaccines in Hungary: the first cold chain monitor study in Europe. Bulletin of the World Health Organization, 1990, 68: 431-439. 8. Expanded Programme of Immunization. Cold chain evaluation (report from Indian Ministry of Health). Weekly epidemiological record, 1988, 63(26): 193- 196. 9. Wassif SM et al. Cluster survey for vaccination cover- age in Sohag governorate, October 1989. Journal of the Egyptian Public Health Association, 1990, 65: 41 1- 426. WHO Bulletin OMS. Vol 74 1996 397