Prevalence and seasonal variation of human Plasmodium infection in Punjab Province, Pakistan

Background: Malaria is the fifth leading cause of death worldwide. Pakistan is considered as a moderate malaria-endemic country but still, 177 million individuals are at risk of malaria. Roughly 60% of Pakistan’s population, live in malaria-endemic regions. The present study is based upon the survey of various health care centers in ten cities of Northern and Southern Punjab to find out the malarial infection patterns in 2015. The diagnosis, seasonal variations, age and gender-wise distribution of Plasmodium spp. circulating in the study area were also included in the objectives. Methods: The malaria-suspected patients ‘16075’ were enrolled for malaria diagnosis using microscopy, out of which 925 were malaria positive which were processed for molecular analysis using nested PCR. The 18S rRNA genes of Plasmodium species were amplified, sequenced, blast and phylogenetic tree was constructed based on sequences using online integrated tool MEGA7. Results: The 364 cases recruited from Northern Punjab with highest incidence in Rawalpindi (25.5%) and lowest in Chakwal (15.9%). From Southern Punjab561 cases were observed Rajanpur (21.4%) maximum and lowest from Multan and Rahim Yar Khan (18%). The slide positivity rate, annual parasite incidence, and annual blood examination rates were 5.7 per 1000 population, 0.1% and 0.2% respectively. The only P. vivax (66.7%), P. falciparum (23.7%) and mixed infection by these two species (9.6%) were diagnosed. The same trend (P. vivax>P. falciparum > mixed infection) in species identification and prevalence was confirmed from molecular analysis. However, the prevalence of malaria was higher in Southern Punjab (5.5%) as compared to the Northern Punjab (4.0%). The overall prevalence of treatment-seeking patients in all recruited cities of Punjab was 4.9%. The age-group of 1-20 and males among genders were more affected by malaria. The comparison of different seasons showed that the malaria infection was at a peak in Summer and post-monsoon. Conclusion: The prevalence of malaria was high in the flood infected rural areas of Southern Punjab, Summer and post monsoon The low age grouped persons and

annually. World is currently not on track to achieve the milestones of 2020 of WHO Global Technical Strategy (GTS) of malaria 2016-2030. The mile stone of 2020 is to reduce malaria death and disease by 40%. There is a continuous increase in malaria cases from 2015 to 2018.The number of malaria cases globally increases as 214, 217 and 219 million from 2015, 2016 and 2017 respectively (1,2,3) In Pakistan 3.5 million suspected and confirmed malaria cases are reported each year. The history of malaria encapsulates our failure to combat global epidemics as, yet it is the leading threat to public health, economic growth and development in many countries [4]. Malaria is caused by five Plasmodium species in human; P. vivax, P. falciparum, P. ovale, P. malaria and P. Knowlesi (zoonotic infection). Regardless of massive and costly malarial control measurements have been taken over many decades but still, it has re-emerged as a serious health problem in Asia. Sixty percent population of Pakistan lives in malaria-endemic regions. Plasmodium vivax contributes 81.3%, P. falciparum 14.7%, and mixed species 4% in the malaria burden of Pakistan [2]. As reported by WHO in 2017 and 2018 [3][4], Pakistan is among the six WHO Eastern Mediterranean region countries with high malaria transmission and about 100% of the population living at risk. The endemicity of malaria varies in different provinces and even in different cities having variable climates. The cumulative API in all over Pakistan in 2017 was 1.8. Province wise breakdown indicates that during 2017 highest numbers of cases were reported from Khyber Pakhtunkhwa 30%, Sindh 26.5%, Federally Administered Tribal Area (FATA) 21.9%, Baluchistan 20.5% and Punjab with the least epidemiology of 1.1% [2].
The endemicity of malaria varies in Punjab being the land of five rivers Sutlej, Chenab, Beas, Ravi, and Jhelum generate a major breeding ground for malaria vector. Punjab is the most populous province of Pakistan with an estimated population of 101,391,000 according to the 2015 Census of Pakistan. It experiences all four types of seasons; Spring (March to May), Summer (June to September), Autumn (October to November) and Winter (December to February). The onset of the Southwest monsoon in Punjab, most likely takes place from the month of June till September but the weather pattern in Pakistan has been irregular since the last few decades. Spring monsoon has either missed or has caused heavy rain over the area that result in floods. The month of June and July are extremely hot. This domineering heat is interrupted by the rainy season in August. The hardest part of the summer season is then over, but cooler weather does not come until late October [5].
Punjab's plains are low-lying, wet and often swamped. Malaria is seasonal and unpredictable in this province and its epidemics repeated at about 8-year intervals. Variation in malaria transmission from year to year is due to the floods in the Southern Punjab and heavy rainfall during monsoon in Northern Punjab. However, the repeated changes in climate patterns are linked with variation in the malaria transmission pattern [6].
Malaria is more prevalent in rural areas due to the low socioeconomic conditions [7]. The prevalence of P. falciparum is high among the Afghan refugees than the local population. Several epidemiological studies have revealed that there was a 24 to 36% increase in the malaria prevalence of Pakistan due to the influx of Afghan refugees [7][8]. The transmission of malaria is seasonal in Pakistan, and a gradual increase in cases can be noted after the July to August monsoon [9]. The P. vivax infection is restrained to two peaks per year, the main peak was in late Spring as a result relapses of earlier infections and additional peaks occurred in Summer and autumn by recent transmissions [1]. In contrast to P. vivax, P. falciparum malaria showed an increasing correlation with August-September temperatures [10]. The wide irrigation network throughout the country, agricultural practices, and monsoon rains provide a favorable environment for the development and growth of malaria vectors [11].
The National Malaria Control Program has reported a six-fold increase in P. falciparum infection in the last decade. The increase of P. falciparum infection across the country can be associated to chloroquine drug resistance [12] also in this part of the world temperature in Autumn is warmer that increase the transmission rate and due to inappropriate measures of vector control activities [13].
The malaria epidemiology is influenced by environmental factors and socioeconomic conditions that supports the vector development and in turn, enhances the parasite and host relationship [14].
Besides these, various other factors such as urbanization, exponential population growth, migration of Afghan refugees, and environmental changes due to excessive monsoon rains, floods, and extensive irrigation projects also favors malarial parasite transmission [1,15].
The correct diagnosis of Plasmodium species is necessary for its treatment and drug formulation. The primary method is microscopy however the most advanced, proficient, sensitive and precise method for the detection of Plasmodium species is molecular analysis using Polymerase chain reaction (PCR).
The PCR analysis can identify Plasmodium species and mixed infections even at low parasitemia [16][17]. It can also be used to find drug resistance and follow-up therapeutic response [18]. The present study is based upon a complete overview of malaria regarding age, gender and seasonal variations in the two opposite zones of Punjab contributing ten cities. The Plasmodium species are also identified circulating in the studied area and their individual prevalence is calculated statistically It will be a great contribution in the epidemiology of malaria in Pakistan and helpful in further malaria control strategies especially in educating people and drug formulation.

Study area
The study areas covered of 10 cities of Punjab from approximately two opposite zones i.e. Northern

Sample Collection
This study has been approved and assigned a Protocol (BEC-FBS-QAU-14) by the Bio-Ethical Committee (BEC) of the Quaid-i-Azam University of Islamabad, Pakistan. A survey was conducted from January to December 2015 based upon 16075 people of the study area. Total 925 microscopically positive malaria patient's blood samples were collected from different hospitals and clinics after taking verbal and written consent from patients/guardians while 15150 were malaria negative cases.
The venous blood (3 mL) was collected in EDTA vacutainers for further molecular analysis.

Demographic investigation of Malarial Patients
The Performa was designed in order to gather further information about the patient's age, sex, locality. Slide Positivity Rate (SPR), Annual Parasite Incidence (API) and Annual Blood Examination Rate (ABER) were calculated by different formulas [19]. The prevalence based on molecular diagnosis was calculated according to Khattak et al. [20].

Molecular Analysis
Blood samples collected at different sites were delivered to Quaid-i-Azam University Parasitology lab in the cold chain. The DNA was extracted by using the Gene Jet Genomic DNA Purification Kit (Thermo Scientific, (EU (Lithuania), according to the manufacturer's instructions and then stored at -20 °C.
Human malarial parasitic DNA was detected through nested polymerase chain reaction (PCR) high sensitive detection method was adopted from Snounou et al. [21] using small subunit (SSU) ribosomal RNA (rRNA) gene for Plasmodium genus and species ( P. vivax P. falciparum, P. malaria, and P. ovale) detection.
The amplification of 18S rRNA gene was carried out using the primers and nested cycling conditions reported by Snounou et al. [21]. In a 1 st round nested-PCR recipe, 50 μL master mix was used containing; PCR water 32.5 μL, phosphate buffer 5 μL, MgCl 2 4 μL, dNTPs 1 μL, 2 µL of each forward and reverse primer, Taq polymerase 0.5 μL and 3 μL DNA template. For the 2 nd round, 4 μL of the 1 st round PCR product was used as a template with the same quantity of master mix as mentioned in the 1 st round. A plasmid containing DNA from Plasmodium species were obtained from MR4 (Malaria Research and Reference Reagent Resource Center) for the amplification of all five Plasmodium species. Few malaria positive samples were also collected from different sentinel sites of the Directorate of Malaria Control Program for the parasitic DNA amplification. Healthy blood samples were used as a negative control. The visualization of amplified PCR products was conducted by electrophoresis using 1.5-2.0% agarose gel stained with ethidium bromide and visualized under UVtrans-illuminator (ExtraGene®, USA). The smear free amplified PCR products were purified using a gene jet PCR purification kit (Thermo Fisher Scientific, EU Lithuania) (cat# K0701) and sent to Korea (macrogen Inc.) for sequencing. The evolutionary tree was constructed using Mega 7 software.

Statistical analysis
The count of malarial incidences was analyzed in relation to the age and gender of patients and the season of prevalence. Initial data exploration revealed that count data is zero-inflated and over-dispersed (mean = 9.64 was much lower than variance = 310.31). We, therefore, tested the following three models in R software [22]: M1: Zero-inflated negative binomial regression with random effect of season Zeroinfl(incidences ~ age + gender + species+ 1 | season, data = Disease, dist = "negbin") M2: Zero-inflated negative binomial regression without random effect Zeroinfl(incidences ~ age + gender + species, data = Disease, dist = "negbin")) M3: Simple negative binomial regression glm.nb(incidences ~ age + gender + species + season, data = Disease) For comparison of the models, the Vuong test was used, which suggested that the negative binomial model was a significant improvement (p = 1.1672e-06) over a zero-inflated negative binomial model. Therefore, the results of the negative binomial model are used in this study. Data visualization was performed using the 'ggplot2' package in the R program.
Cohen 'Kappa was calculated to measure the level of agreement between two diagnostic tests i.e.

Area wise distribution and incidence
Out of 16075 entire suspected cases, 925 were positive and 15150 were negative. The overall slide positivity rate (SPR) was 5.7% in all recruited cities of Punjab however; the SPR was higher in Southern Punjab (6.1%) as compared to the Northern Punjab (5.3%). The SPR within the recruited cities of Northern Punjab was highest in Jhelum lowest in Chakwal i.e. 6.8% and 3.1% respectively. In contrast among observed cities of Southern Punjab, the SPR was maximum in Rajanpur  Table 1).

Seasonal variations in malaria incidence
The negative binomial regression analysis (Table 2) indicated a strong effect of season on malarial incidences in Punjab. Autumn (used as a control in the analysis) had a higher rate of incidences as compared to Spring and Winter with expected incidence (on a log scale) of -0.5671 and -2.1374, respectively. The Summer was the peak season with expected log (incidence) of 0.3737 higher than that of Autumn holding other variables constant (Fig. 2).

Effect of age and gender on malaria incidence
Age was a strong indicator of malaria prevalence, the young people (<20 years) were most susceptible to the disease, and risk declined gradually in older ages ( Table 2). For example, the incidence in the age group 21-40 was -0.1271 lower (on a log scale) than that of the age group of 1-20 years holding other variables constant. Similarly, incidences in age groups 41-60 and above 60 years were -0.9392 and -2.4390 lower, respectively, then that in young people (Table 2, Fig. 3).
Males had an expected log (incidence) of 0.8466 higher than that of females holding other variables constant. The P. vivax had an expected log (incidence) of 1.0308 higher than that of P. falciparum, however, mix infections are -0.8562 lower than P. falciparum (Table 2, Fig. 3). In conclusion, age, gender, species and season are significant predictors of malaria incidences in Punjab.
However, in comparison, the overall incidence was high in the Southern Punjab as compared to Northern Punjab.
The coincidental adjustment of kappa statistics specified that overall agreement in the presence or absence of Plasmodium species was good (Kappa = 0.79). However, for the detection of P. vivax the agreement between microscopy and PCR was fair (Kappa = 0.38). But for P. falciparum and mixed infection, it was moderate (Kappa = 0.53, 0.59) respectively.

Molecular Epidemiology
The overall prevalence of treatment-seeking patients in all recruited cities of Punjab was 4.9%.
However, the comparison of Northern and Southern Punjab indicated that the prevalence was higher in Southern Punjab (5.5%) as compared to the Northern Punjab (4.0%). Within Northern Punjab prevalence was higher in Rawalpindi i.e. 5.0% followed by Gujrat 4.9%. The malaria prevalence was lowest in Chakwal i.e. 2.3%. Among the studied cities of southern Punjab, the prevalence followed the pattern Rajanpur (6.7%) > Bahawalpur (5.3%) > Rahim Yar Khan

Phylogenetic analysis
A phylogenetic tree was constructed based upon sequenced results of both P. vivax and P. falciparum taken from Northern and Southern Punjab two from each site P. vivax and one of P. falciparum (Fig.   5). The tree inferred two distinct clades. The P. vivax isolates from Northern and Southern Punjab,

Discussion
Malaria is a serious health problem in Pakistan. Its cases vary significantly in different areas and cannot be assessed accurately due to lacks different information [23]. The parasite species of malaria also have unequal distribution throughout Pakistan and their prevalence changes in seasons.
Therefore, it has been difficult to assess the accurate prevalence of malaria infection in Pakistan. The occurrence of Plasmodium infection can be effectively reduced using active and passive diagnosis [20]. The current study implies on the survey of malaria from ten endemic cities of Punjab, Pakistan is experiencing all four seasons with extreme weather conditions. The WHO in 2014 [24] reported that about 3.3 billion people in 97 countries are at risk of malaria, and 1.2 billion are at high risk. Greater than 1 case of malaria per 1000 population each year was observed. World Health Organization [25] reported about one million microscopically positive malaria cases in 2010 from the Eastern Mediterranean region out of the 22% were from Pakistan. The definite and accurate estimate of Plasmodium infection can also be useful in scaling up malaria surveillance and control measures in Pakistan [13]. In the present study total, 16075 suspected cases of malaria were ascertained.
However, 925 were microscopically positive. Concerning spatiotemporal distribution, more cases were recruited from Northern Punjab as compared to Southern Punjab. The Rawalpindi from the Northern zone and Rajanpur from the Southern zone have the highest malaria cases among all recruited cases.
The overall (SPR) in two zones of Punjab was 5.7%. However, the SPR was high in Southern Punjab 6.1% > 5.3% as compared to Northern Punjab. Mahmood  The incident of malaria began to increase in the spring season i.e. from March and April. The seasonal variation such as summer monsoon season (from June to September) showed that highest malaria incidence due to highly favorable environmental conditions for mosquitoes breeding However, towards November to February (coldest season) lowest malaria cases occur due to a change in climate, such change in temperature and rainfall patterns unstable the vector flux. The age wise distribution of malaria was also evaluated in the present study. The age of all recruited cases in both genders was ranged between 1 to 78 years. The high incidence was observed among the age group of 1-20 >21-41 than among 41-60-year age groups. However, the distribution of malaria parasites was low in high age groups. It indicates that the 1-20 age group is highly infected due to low acquired immunity and exposure to vector because of higher non-resting outdoor activities in this age group.
The gender wise distribution indicated that Plasmodium infection was dominant in males than females in both zones of Punjab. In the present study, 71% male and 39% female were infected. The female in the Southern Punjab (30.5%) was comparatively high as compared to the Northern Punjab (27.5%).
The possible reason may be the role of males in agricultural activities. Khattak et al. [20] reported that males were 64% and females were 36% infected in Punjab. Different reports from different areas of Pakistan showed the males being more affected by Malaria than females [27][28], which are comparable to the present findings. This gender-wise variance in malaria transmission is probably due to the socioeconomic norms of Pakistan like the low participation of females in agricultural activities and little access of females to the health care centers and hospitals. falciparum. However, the present studies revealed P. vivax as 66.7% and P. falciparum, 23.7% and mixed infection (P. vivax and P. falciparum) was found to be 9.6% by microscopy. The molecular analysis confirmed the presence of the same species with independent and mixed infection although there was a difference in prevalence e.g. P. vivax: 53.4%, P. falciparum:16.70% and mixed infection: 12.9 %. However, the 139 (15.2%) microscopically positive samples did not amplify with the PCR analysis that may be due to the false positive results of microscopy or may be due to the DNA damaged by anticoagulant used in EDTA tubes. The high prevalence of P. falciparum in contrast to P. vivax (65% vs 35%) among 100 children positive for malaria in a university hospital has been recorded [31]. Mahmood et al. [26] reported a high percentage of P. falciparum (88.5%) as compared to P. vivax (9%) out of 348 recruited malarial patients in Karachi, Pakistan, whereas, in Multan, P. vivax and P. falciparum were observed in a percentage ratio of 60.5:37.2 respectively [32]. The present findings are also in accordance with and following the same pattern in the distribution of Plasmodium species from Southern to Northern areas of Pakistan P. vivax infection is becoming higher whereas, P. falciparum is going to decrease may be due to variation in temperature.
With respect to socioeconomic norms, WHO (2010) [33] reported that rural areas of Pakistan were more infected with malaria The present results also revealed the more prevalence of malaria in Southern Punjab cities due to low development, lack of access to health care facilities, use of presumptive treatment, as compared to Northern Punjab cities included in the study area. The environmental factors such as floods after unprecedented monsoon rains increase site areas for mosquito vector growth thus increasing malaria incidences. All cities of Southern Punjab indicated that P. vivax had a high prevalence except Bahawalpur showed P. falciparum as predominant species.
Similarly, in Northern Punjab P. vivax had the highest prevalence and Chakwal was accounted for the highest mixed infection. The recent flee of IDPs (Internal displacement people) from high malaria prevalence areas such as FATA [34][35][36] and Swat [13] to Northern Punjab due to war operations against terrorism has contributed to the high prevalence of P. vivax in Northern Punjab. The phylogenetic kinship of P. vivax and P. falciparum isolates is rather not restricted to one region but seems to be widespread across different geographical regions of the world. The isolates of P.
vivax from Northern and Southern Punjab showed homology to the P. vivax isolates of Yamen and India. All isolates of P. falciparum were clustered together and showed homology of P.
falciparum isolates from Yunnan, Africa, and India. Similar origin of P. falciparum was observed by Joy et al. [37]. However, P. vivax showed a more diverse population than P. falciparum. A significant rise in temperature and humidity result in an increase of vector breeding rate and the vectoral capacity that may enhance the Plasmodium infection and its re-emergence [36,38]. The microscopic observation is a conventional, cheaper and less time-consuming method for the detection of Plasmodium Species. However, its accuracy of the results mostly depends upon the experience of technicians along with other factors like slide staining pattern, parasite level in the blood and microscope condition also influence the results. PCR is used for the most accurate diagnosis of all the five species of Plasmodium, but it is expensive than microscopy. The epidemiological study of malaria conducted in the Orakzai agency showed a higher positive result of microscopy as compared to PCR [35][36]. Shahzad et al.in 2013[39] conducted a molecular study for the diagnosis of malaria and found that the PCR based diagnosis is more sensitive, specific and accurate as compared to microscopy that is standard for routine laboratory diagnosis of malaria. In the case of low parasite density and mixed infections, microscopy is not a sensitive test. According to Steenkeste et al. in 2009 [40], PCR based diagnosis is the best option for the large-scale epidemiological studies especially for the diagnosis of mixed infections.

Conclusion
The malaria control remains a great challenge in developing countries like Pakistan due to agricultural activities; prolong the monsoon season and frequent floods. The relationship between seasonality and Plasmodium occurrences were studied. This study exposed that malarial endemicity was highest in Summer and Post-monsoon season might be because of rice paddies and sugarcane cultivation are at peak in this season, which increases the rate of mosquitoes breeding. Along with the effect of seasonal variations on malaria incidence, other contributing factors which may increase malaria endemicity are like misdiagnosis by untrained microscope technician, presumptive treatments, extensive agriculture activities, improper waste management, inadequate health care facilities in remote areas, low socioeconomic conditions and lack of malaria control intervention strategies. The overall incidence rate of malaria in Southern Punjab found to be high as compared to Northern Punjab may be due to the flood in Southern Punjab. P. vivax was found to be more prevalent than P. Additional files and the availability of data Additional files Graphical abstract and statistical model script are provided along with the manuscript.
While the availability of prevalence raw data will be provided by the corresponding author on reasonable request.

Funding
This work was supported by Quaid-i-Azam University, Islamabad. Pakistan.
Ethics approval and consent to participate

Consent for publication
Not applicable.

Competing interest
The authors have no competing interests.

Declaration
The data presented in this study contain confidential and private information about the patients and their locations. To share this information is ethically not allowed. However, the analyzed data without patient's personal information and hospital and private labs can be requested from the corresponding author if required.        Phylogenetic relationships of P. vivax and P. falciparum isolates from Northern and Southern Punjab. Plasmodium vivax clustered into four and P. falciparum clustered into two subclusters within the main clade. The neighbor-joining method was applied to infer evolutionary history. The branch length = 6.06796875 was shown and the percentage of replicating trees was shown next to the branches. The p-distance method was used to evaluate evolutionary distance and it was in the units of the number of base differences per site. The analysis involved 21 nucleotides DNA sequences.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.