Effect of Temperature and Distance on the Viral Outcome of Clinical Specimens From Acute Flaccid Paralysis Cases in Nigeria

Abstract

Aim

The effect of cold box temperature and distance on virus titers, poliovirus isolation rate, and appearance of orphan polioviruses was investigated.

Methods

Cold boxes with stools were randomly selected and examined for internal temperature over a 7‐month period. After virus isolation in Rhabdomyosarcoma (RD) cell line RDand L20B, titer calculations and intratypic differentiation were done on isolates. Sequencing and molecular studies were done on the isolates periodically in the order of arrival in the laboratory for a period of 30 months.

Results

Seventy‐one (51.1%) boxes had the temperature range of 1 –4°C, 53 (38.1%) had 4.5 –8°C,. while 15 (10.8%) had temperature between 8.5°C and 17.0°C.

Poliovirus was isolated from 24 (8.6%) specimens made up of 13 wild 1 and 2 and 11 Sabins 1, 2, 3 with titers between 10^1.8 and 10^5.4 TCID ₅₀/100 μl. Temperature and titer were inversely proportional and statistically significant. (r = −0.83, P < 0.05). Distance to laboratory was not significantly related (r = −0.025) to temperature when appropriate cold box temperature was maintained. Of the 18,188 acute flaccid paralysis (AFP) specimens received in the laboratory between June 2008 and December 2010, 1,752 poliovirus isolates (9.6%) consisting of 480 wild and 82 orphans were found. A positive correlation between the distance and orphan viruses (r = 0.425; P = 0.027) was observed.

Conclusion

While poliovirus titer depends on the inside temperature of the cold box, distance to the laboratory was found to be a predisposing factor to the appearance of orphan viruses. J. Clin. Lab. Anal. 26:254‐261, 2012. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Poliomyelitis is caused by poliovirus, a nonenveloped single‐stranded RNA virus with human as the only natural host, but antibodies to the virus can be detected from primate monkeys (1. Because of this biological characteristic of single natural host and no serious reservoir problem in other animal host, it was easy to target the virus for eradication. In 1988, the World Health Assembly (WHA) established a resolution to eradicate poliomyelitis globally by the year 2,000 2. Implementation of the program was hinged on mass immunization, field surveillance, and laboratory testing and identification. In a network of accredited laboratories, two stool specimens are collected for each acute flaccid paralysis (AFP) case and transported to the laboratory where the samples are tested for the presence of poliovirus. Following the aggressive use of the live attenuated oral polio vaccine (OPV), the number of poliomyelitis cases has been reduced from 350,000 cases in 175 countries in 1988 to only 1,655 cases in 2008 3, 4. The virus is now limited to a few countries of which Nigeria, Afghanistan, India, and Pakistan remain the major reservoirs still transmitting the virus 3, 4.

Stool is the preferred specimen of choice for virus isolation and identification 5. The ability of the laboratory to isolate and identify the virus from clinical samples depends to a large extent on the quality of stool brought to the laboratory. To meet the adequate conditions of good stool specimen, a reverse‐cold chain system containing padded cold box and ice packs was devised to transport stool specimens to the laboratories. However, because of unavoidable challenges such as long distance between the field and the laboratory, poor mode of transportation, unforeseen delay in the course of the journey, negligence of field officer on duty, and interrupted power supply, the reverse‐cold chain is often broken, making the temperature inside the cold box higher than the required temperature.

Break in cold chain can lead to failure to isolate the virus from a positive sample 6, 7 thus supplying wrong result to the program. This in turn can later give rise to the appearance of orphan viruses as a result of a continuous undetected circulation of the virus 8, 9.

In Nigeria, there are two World Health Organization (WHO) polio laboratories; one of them is located in Ibadan in the Southwestern part. This laboratory serves 26 of the 36 states, including the Federal Capital Territory (FCT) with the shortest distance to the lab of about 4 km and farthest distance of about 1,036 km. The second laboratory is located in Maiduguri in the Northeastern part and serves the remaining 10 states. The shortest distance to this laboratory is about 20 km and farthest distance of about 876 km. This study, which was conducted at the Ibadan Laboratory, was aimed at evaluating the condition of the cold‐chain system as well as determining the polio isolation rates, the titers of poliovirus isolates, and any probable relationship with the appearance of orphan viruses from the affected states.

MATERIALS AND METHODS

Sample Collection and Transportation

Two stool specimens collected at 24‐hour interval between June and December 2008 were gathered from clinically diagnosed AFP cases by the Disease Surveillance Notification Officer of the affected states and transported to the laboratory in the WHO‐recommended cold box. The method of transportation of samples to the laboratory was the public transport system originating from the states. Depending on the distance to the laboratory, there might be one or two disembarkments before arrival in the laboratory. Neither the program, nor the individual conveying the samples to the laboratory had any influence on the mode of transportation. The date of sample collection and arrival in the laboratory for each sample were observed and recorded.

Temperature Monitoring of Cold Box and Virus Isolation

Cold box containing stool samples were randomly selected as they arrived in the laboratory. The internal temperature of the cold box and other stool‐adequacy conditions were observed and recorded. This included the quantity and quality of the stool, accurate documentation, lack of desiccation, and evidence of leakage.

Stool samples preparation and virus isolation in Rhabdomyosarcoma (RD) and L20B cells were done as described in the polio laboratory manual 7. Cells showing characteristic poliovirus cytopathic effect (CPE) were removed and stored at −20°C till virus titration. Isolates were titrated and virus titer was calculated using the Karber formula 10.

Intratypic Differentiation of Isolates

Poliovirus positive samples were differentiated into wild or Sabin pathotypes by the ELISA and the reverse‐transcriptase polymerase chain reaction, as previously described 7.

Molecular Epidemiological Study

In an attempt to establish any genetic changes in the nucleotide sequences and possibilities of appearance of orphan viruses, all the suspected poliovirus isolates from the 26 states under study were followed up for a period of 30 months from July 2008 to December 2010. These positive samples were further analyzed at the Polio and Enterovirus Laboratory of the Centers for Disease Control and Prevention for sequencing and molecular study to determine any changes in genotype or variation in the nucleotide sequences of the viruses.

Nucleic Acid Sequence Analysis

Sequencing of the Virus protein 1(VP1) region of poliovirus isolates was performed as described by Kilpatrick et al. 11. Genetic analysis of the viruses to determine orphan status was performed by first ordering each viral sequence to be analyzed by the case‐onset date obtained from patient epidemiological data. A pairwise nucleotide comparison of the ordered sequences to a database of wild‐ and vaccine‐derived poliovirus VP1 sequences was accomplished using the Needleman‐Wunsch algorithm 12. Orphan status was defined as any virus having ≤98.5% nucleotide identity with its closest match in the database. Evolution history of the poliovirus sequences was inferred using the Neighbor‐Joining method 13 with evolutionary distances computed using the Kimura 2‐parameter method 14. Dendrograms were constructed using the MATLAB bioinformatics toolbox (ver 2010 a, MathWorks, Natick, MA, USA).

Statistical Analysis

Statistical Package for Social Sciences (SPSS 16.0) and PASW 18 statistics were used to analyze the data. Pearson's correlation coefficient of variables was used to determine relationship and significance between cold box temperatures, as well as the distribution of orphan viruses by distance to the laboratory and cold chain maintenance. Differences were considered statistically significant at P < 0.05. Regression line was fitted for dependent (titer) variable and independent (temperature) variable. A relationship was computed to predict the value of titer at 0°C.

RESULTS

A total of 278 specimens were investigated during the 7‐month period of study. The various cold box temperature regimes recorded in the study were categorized into three with interval of four starting from 1.0 –4.0°C, 4.5 –8.0°C, 8.5°C, and above. The interval of 4°C was selected because it has been reported that at this, the deviation loss of titer seems inevitable 15, 16. Seventy‐one boxes (51.1%) had temperature ranging between 1 and 4°C, while 53 boxes (38.1%) had temperature ranging between 4.5 and 8°C. Fifteen boxes (10.8%) had temperature ranging between 8.5 and 17.0°C. The most frequently encountered temperature was 3.5°C.

The minimum average temperature was 2.5°C (Rivers State) while the maximum was 17.0°C (Ebonyi State). Poliovirus was isolated from eight of the 26 states and the FCT. Only 24 specimens made up of 13 wild 1 and 3 and 11 Sabins 1, 2, 3 tested positive for poliovirus. (Table 1) Pearson correlation shows a low negative correlation coefficient between approximate distance covered and the average temperature regime (r = −0.025).

Table 1.

Profile of AFPsamples received at the Ibadan Polio laboratory between June and December, 2008

State	Distance to laboratory	No. of stool specimen	Average cold box temp. (°C)	No. of isolates
ABS	551	8	4.1	0
AKS	718	4	6.0	0
ANS	503	8	5.9	0
BNS	662	10	2.7	0
BYS	711	6	5.7	0
CRS	727	8	5.6	0
DTS	388	6	4.1	0
EBS	486	2	17.0	0
EDS	291	8	6.4	0
EKS	262	6	5.2	0
ENS	539	12	6.1	0
FCT	659	8	3.8	2
IMS	527	4	4.2	2
KBS	804	12	4.0	4
KDS	759	18	3.4	2
KGS	648	12	4.9	0
KWS	159	8	3.8	0
LAS	147	18	4.4	0
NAS	703	8	4.1	0
NIS	596	22	4.3	4
ODS	206	6	2.8	0
OGS	77	8	5.1	0
OSS	88	12	4.3	0
OYS	4	16	6.0	2
RVS	625	8	2.5	0
SOS	892	16	5.3	0
ZAM	816	24	5.7	6
Total	‐	278	‐	24

Table 2 shows the number of poliovirus isolates from states with positive samples with their corresponding titers. The highest average virus titer was observed in samples from Kaduna State with a distance of about 759 km from the laboratory and an average cold box temperature of 3.4°C. The lowest titer was observed in samples from Zamfara State with a distance of 816 km from the laboratory and an average cold box temperature of 6.3°C. Pearson correlation showed that titer and cold box temperature regime were significantly related as revealed by high negative correlation coefficient (r = −0.83). Regression line was fitted for dependent variable (titer) and independent variable (temperature) to establish a relationship. Maximum temperature of 16°C was predicted when titer was zero. Temperature and titer were inversely proportional and statistically significant (r = −0.83, P < 0.05). The titer of viruses in the cold boxes with temperature of 16°C and above was zero. Distance between the place of specimen collection and laboratory was not significantly related (r = −0.025) to the temperature when the appropriate cold box temperature was maintained.

Table 2.

Showing distance from laboratory, cold box temperature, virus isolates with their corresponding titers

State	Distance (Km)	No. of specimen	No. of isolate	Average cold box temp. (°C)	Titer range TCID50
Imo	527	4	2	7	2.5–2.5
Kaduna	759	18	2	3.4	4.8–5.4
Kebbi	804	12	4	3.5	3.3–3.3
Niger	596	22	4	3.0	3.5–4.8
Oyo	2	16	2	6.5	3.2–3.2
Sokoto	892	16	2	5.5	3.2–3.3
Zamfara	816	24	6	6.3	1.8–4.5
FCT	659	8	2	6.5	2.7–2.8
Total		120	24

A total of 18,188 AFP specimens were sent to the laboratory between June 2008 and December 2010. Between 85and 87% of all the samples were sent to the laboratory within 72 h after collection. There was no significant correlation between the time of collection of sample or arrival in the laboratory and the observed titers of viruses and appearance of orphan viruses (r = −0.025; P < 0.05). Poliovirus was isolated from 1,752 samples giving a polio isolation rate of 9.6%. Four‐hundred and eighty were wild polioviruses, of which 82 were orphan viruses (Table 3, Fig. 1). The orphan viruses had percentage homology between 96.9 and 98.5% depending on how long they have been circulating undetected. They were mostly related to viruses circulating within the state or neighboring state and in some rare occasions, with neighboring country like the Niger Republic. The highest number of orphan was in 2008 (Figs. 1 & 2). However, the highest percentage of orphan viruses in relation to the number of wild viruses (36%) was in 2010 where there were a total of nine orphan viruses from the 25 polioviruses for the whole year (Table 3). On all occasions, the highest number of orphan viruses was from Zamfara State, 816 km from the laboratory (Fig. 1) and an average cold box temperature of 6.3°C from the previous study. A positive correlation between the distance from the site of stool collection to the laboratory and isolation of orphan virus (r = 0.425; P = 0.027) was observed.

Table 3.

Showing the number of viruses from the extended study (June 2008–December 2010) with the number of isolates and orphan viruses

State	Average cold box T°	Distance from lab	No. of specimens			No. of isolates			No. of wild			No. of orphans
			‘08 Jun–Dec	‘09 Jan–Dec	‘10 Jan–Dec	‘08 Jun–Dec	‘09 Jan–Dec	‘10 Jan–Dec	‘08 Jun–Dec	‘09 Jan–Dec	‘10 Jan–Dec	‘08	‘09	’10
ABS	4.1	551	66	144	120	6	28	18	0	2	0	0	0	0
AKS	6.0	718	128	264	200	0	14	43	0	0	0	0	0	0
ANS	5.9	503	92	170	152	0	12	19	0	0	0	0	0	0
BNS	2.7	662	132	288	204	3	33	28	0	10	0	2	0	0
BYS	5.7	711	96	164	154	0	13	3	0	6	0	0	0	0
CRS	5.6	727	92	156	138	3	9	7	0	0	0	0	0	0
DTS	4.1	388	130	330	230	3	40	14	0	14	2	0	1	1
EBS	17.0	486	62	238	153	2	25	23	0	4	0	0	0	0
EDS	6.4	291	159	292	241	0	22	9	0	1	0	0	0	0
EKS	5.2	262	80	100	110	4	6	2	0	0	0	0	0	0
ENS	6.1	539	164	212	276	9	7	18	2	0	0	1	0	0
FCT	3.8	659	70	109	134	14	6	25	13	2	1	2	0	1
IMS	4.2	527	70	188	144	2	7	23	0	0	0	0	0	0
KBS	4.0	804	188	462	304	20	59	28	12	32	6	4	4	4
KDS	3.4	759	296	372	466	61	82	29	44	31	0	5	0	0
KGS	4.9	648	340	410	540	25	27	14	14	11	0	4	0	0
KWS	3.8	159	100	192	210	14	23	16	10	4	0	2	0	0
LAS	4.4	147	318	482	506	5	36	44	6	7	0	0	0	0
NAS	4.1	703	114	228	200	10	40	25	6	17	0	4	0	0
NIS	4.3	596	253	460	474	30	81	35	8	27	0	4	0	0
ODS	2.8	206	46	136	142	2	9	7	2	0	0	0	0	0
OGS	5.1	77	166	276	254	5	45	12	0	28	0	2	0	0
OSS	4.3	88	120	188	210	11	6	14	4	0	0	0	0	0
OYS	6.0	4	140	248	246	18	15	12	8	4	0	3	0	0
RVS	2.5	625	176	206	272	2	19	11	0	0	0	0	0	0
SOS	5.3	892	186	366	320	28	52	39	21	32	7	7	2	1
ZAM	5.7	816	352	380	590	102	85	44	57	26	9	20	6	2
Total			4,136	7,061	6,991	379	801	572	207	24,825		60	13	9

Figure 1.

Geographic distribution of orphan polioviruses.

Figure 2.

Graph showing the number of orphan viruses according to distance of state from the laboratory.

On the other hand, there was no significant correlation (r = −0.47; P = 0.815) between the mean cold chain temperature and the isolation of orphan viruses.

DISCUSSION

The laboratory is one of the three tripods on which the polio eradication stands and its function is to isolate and type the wild polioviruses from AFP cases and use the result to direct immunization and surveillance.

For the laboratory to perform this function successfully, stool must arrive in the laboratory in good condition, one of which is the right temperature. Temperatures between 8 and −20°C are recommended by the WHO for the transportation and storage of stool samples and polio isolates, respectively 6, 7. In this study, the cold box temperature of specimen was monitored from the 26 states and the FCT served by Ibadan WHO Polio Lab. One‐hundred forty‐two specimens representing 51.1% were brought to the laboratory at 1–4°C temperature range. This might have been responsible for the high polio isolation rate observed in the study although virus titers differ. The poliovirus isolation rate observed was 8.6% titer differs. Two samples arrived at 17°C. The result of isolation on these two specimens showed that they were negative. It is not unlikely that these two specimens initially contained some viral particles that were inactivated by the high cold box temperature. Statistical analysis in this study also predicted zero titer at maximum 16°C. The implication of this is that an orphan virus might be detected in the area in the future. However, at the end of this study, no orphan virus was detected in Ebonyi State. The detection of any circulating orphan virus will depend, to an extent, on the sensitivity of the surveillance system in the state. Epidemiologically, orphan viruses are those viruses that, at the point of isolation and identification, have no close ancestral relationship with their immediate circulating strain or serotype. This is represented by the percentage of nucleotide changes between the orphan and their closest match. In this study, the percentage identity between the orphan and their closest matches was between 96.8 and 98.1% (Table 3). Orphan viruses appear because they had earlier been missed or undetected as a result of poor surveillance or low level of virus isolation rate. They are represented in dendrograms by long branches. Because they have been circulating undetected for a long time, there is always a long time interval separating the orphans from their closest match (Fig. 3).

Figure 3.

Dendrogram showing orphan viruses (arrowed)

2006 viruses are shown in BLACK

2007 viruses in BLUE (first line)

2008 viruses (Orphan) in RED (fifth and sixth lines).

Statistical analysis showed that the distance of the place of collection of specimen to the laboratory was not significantly related to the temperature when the appropriate temperature was maintained. This therefore implied that the major factor that might have affected any loss of titer of the virus is the temperature of the cold box rather than the distance to the laboratory.

Although the poliovirus is a nonenvelop virus, when compared with other RNA viruses, it is relatively stable. However, the virus cannot stay long in the environment of adverse temperature. The poliovirus is stable for weeks and days at room temperature, i.e., 4°C 6, 7, but will be inactivated within 60 min at 44°C 17.

The hypothesis of this study is that lower the temperature of the cold boxes, higher the chance of recovering the poliovirus from the stool; while, higher the temperature, lesser is the likelihood of recovering the virus from the stool. A significant P‐value for the distance from laboratory (P = 0.005 < (a = 0.05)) was observed. This implies that the distance from the lab has a significant relationship to the number of isolates retrieved from the specimens and indirect relationship with the appearance of orphan virus. The number of isolates does not significantly depend on the temperature of the cold box or the interaction between distance from laboratory and temperature of cold box (P = 0.804 and P = 0.843).

Although no significant relationship was detected between the distance from the lab and the temperature of the cold box, further study of the between‐subject effects made a distinction between the levels of temperature. If the temperature of the cold box is less than or equal to 4.2°C (i.e., cold), the distance from the laboratory does not play a significant role in the number of isolates retrieved (P = 0.108). However, if the temperature of the cold box is above 4.2°C, then the distance from the lab plays a significant role on the number of isolates retrieved (P = 0.031 < 0.05). This interaction may be partly responsible for the large number of orphan viruses isolated from Zamfara State with cold box temperature of 5.7°C and a distance of about 816 km from the laboratory. Warmer the temperature of the cold box, the greater the role of distance from the laboratory plays on the number of isolates.

Since there was no significant relationship between the distance from laboratory and the temperature of the cold box, this implies that we cannot propose that the cold box gets warmer with greater distance from the laboratory or vice versa. In essence, the distance from the laboratory is not the cause of the cold box's temperature.

Results and observations from this study have shown that other factors apart from distance and cold box temperature may be responsible for the loss of titer, missing of isolates, and the possible appearance of orphan viruses in the samples. These factors may include the efficacy of surveillance 13, sample handling, and time between collection of sample and arrival in the laboratory. However, no significant correlation between the time of collection of sample/arrival in the laboratory and the titers of viruses and appearance of orphan viruses (r = −0.025; P < 0.05) was observed in this study.

A positive correlation between the distance from the site of stool collection to the laboratory and isolation of orphan virus (r = 0.425; P = 0.027) was observed. Cases of individuals who transport samples to the laboratory and only maintain cold chain at the point of delivery to the laboratory have been observed but often not backed up by evidence. On the other hand, there was no significant correlation (r = −0.47; P = 0.815) between the mean cold chain status and the isolation of orphan viruses. Result obtained from this study has therefore shown that warmer the temperature of the cold box, greater the role distance from the laboratory plays on the maintenance of the titer of the virus, the number of isolates retrieved from the specimen, and the likely appearance of orphan viruses. It is therefore recommended that in order to get a greater number of isolates from the collected specimen and maintain higher titer of the virus and less possibility of appearance of orphan viruses, it is important to have the cold box at the right temperature for the total distance from the state to the laboratory.