Malaria Slide Bank to Strengthen and Improve the Quality of Malaria Diagnosis: A National Slide Repository in India

ABSTRACT.

Malaria elimination is one of the top health care priorities in India, necessitating accessible and accurate diagnosis for effective treatment. A malaria slide bank in India is a collection of quality-controlled malaria-positive and -negative slides and is considered a vital asset for quality diagnosis. The collection of blood samples, preparation of blood smears, staining, quality control, molecular characterizations, and slide validation were carried out according to standard operating procedures in accordance with the WHO reference laboratory. The true count and parasite density per microliter were computed in accordance with WHO guidelines. Over 27 months, 48 batches (8,196 slides) were prepared. Overall, the majority of slide batches were Plasmodium vivax (45.9%; 22/48), followed by Plasmodium falciparum (25%; 12/48), malaria-negative infections (25%; 12/48), and mixed infections (4.1%; 2/48). All 48 batches passed internal validation by WHO-certified level-1 microscopists. For a batch, the true count was the median of the validators’ counts (range, 111–280,795 parasites/µL). Except for mixed infections, the PCR results agreed with the verified microscopy results. Malaria slide bank slides would be a valuable tool for quality control, assurance, and microscopist training.

INTRODUCTION

Despite recent notable progress towards the goal of elimination, malaria still poses a serious threat to India’s public health. An estimated 247 million cases of malaria were recorded globally, with the number of malaria deaths estimated at 619,000 in 2021.¹ The WHO global technical strategy for malaria recommends that populations must have quick and easy access to accurate malaria diagnosis and efficient treatment to accomplish the elimination goal.¹ Accurate diagnosis by microscopy or rapid diagnostic test is mandatory before initiation of antimalarial medications and is the prerequisite for certifying a country as malaria-free by the WHO.

Despite the limitations of both diagnostic methods, microscopy is the “gold standard” method for malaria diagnosis.^2,3 The WHO has recommended strengthening the nation’s quality-assured diagnostic capabilities, including the establishment of a national malaria slide bank (MSB), an external competence assessment of malaria microscopists, and training of malaria microscopists so that they can act as potential facilitators in conducting further training.^4,5

The MSB also seeks to improve the malaria quality assurance program of the national malaria program of the country by providing quality-controlled (QC) slide panels for microscopist training in a laboratory setting. The MSB intends to support activities like basic training and refresher training for malaria microscopy, which can later be used for conducting the National Competency Assessment of Malaria Microscopists (NCAMM) after external validation. The maintenance of an MSB requires funds, dedicated staff, equipment, supply space, and other logistics, as well as strict adherence to the processes. This article describes the steps involved in establishing a national MSB in India at the Indian Council of Medical Research–National Institute of Malaria Research (ICMR-NIMR) in New Delhi for public health improvement.

MATERIALS AND METHODS

The ICMR-NIMR has established the MSB to provide QC slide panels to serve as a reference for malaria diagnosis in India. The WHO Malaria Microscopy Quality Assurance (QA) Manual was referenced for more information pertaining to MSB requisites and development procedures.⁴ Blood samples were collected for the MSB from both malaria-positive and -negative patients from different sites (Government Medical College, Jagdalpur; Darbha Community Health Center in the Bastar District of Chhattisgarh; Dhunseri, Primary Health Center, Udalguri, Assam; and ICMR-NIMR fever clinic, New Delhi).

Screening of patients.

Bivalent rapid diagnostic tests (SD Bioline malaria Ag P.f./P.v. test) and blood smear examinations were conducted to screen febrile patients. The blood sample was collected in an ethylenediaminetetraacetic acid Vacutainer tube and then labeled with a unique identification number, date, and time of collection in accordance with the standard operating procedure.⁴ Blood from a malaria-free healthy donor who consented to provide 3–5 mL of blood was collected for malaria-negative blood slides. For each positive and negative blood sample, 200 thick and thin smears were prepared within an hour after collection and stained with a fast-staining method using 10% Giemsa stain for microscopic analysis.

Blood slide preparation and Giemsa staining.

A slide template with a premarked circle and fine lines on white paper was used to support the presentation of thick and thin blood smears. For a thick smear, 6 µL of blood was applied to the center of each circle allotted on the slide template, and the smear was prepared. The thin smear was prepared using 2 µL of blood and placed on the middle portion of the line (next to the circle) allotted for the thin smears. Smears were allowed to air-dry overnight and were protected from dust.⁴

Thin films were fixed by dipping them in a coupling jar for 3–5 seconds, and the fixed smears were left to air-dry completely (approximately 2 minutes) by placing the slides on a flat surface. A 100-slide-capacity rack/holder was used for batch staining using freshly prepared 3% Giemsa solution in pH 7.2 phosphate buffer. Giemsa stain quality was regularly checked for each batch. The standard time for mass staining was optimized at 45 minutes. However, the duration depends on the staining of the parasites and white blood cells (WBC). To estimate the exact timing, four QC slides were placed in each batch and checked at different time intervals. After staining, the slides were washed under slow-running water to prevent the washing out of the smears. The smears were dried properly and mounted with a coverslip using dibutylphthalate polystyrene xylene mountant and then labeled.⁴ (See Table 1 for methods of assigning a unique identification number.)

Table 1.

Method of assigning a unique donor ID number in accordance with an agreed scheme

Digits in ID Number	Indication
First 2 digits	Site code (country)
Next 2 digits	Year of collection
Last 3 digits	Unique donor ID number
Sample ID number 91-20-001	91 = country code for India; 20 = round 1, year 2020; 001 = unique donor ID number

ID = identification.

Blood spot collection.

The Whatman filter paper was prepared using 125 µL of blood and dried for 1 hour at room temperature before placement in a polyethylene zip-lock bag along with desiccant for further DNA extraction, and then it was stored at –20°C.

Molecular analysis.

DNA was extracted according to the manufacturer’s instructions from the dried blood spot (DBS). Four different malaria parasite species (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale) were identified using a species-specific nested PCR method targeting the 18S ribosomal RNA gene for molecular diagnosis.⁶

Quality control analysis.

All stained slides underwent internal quality control (IQC) by technicians, and slides that did not pass IQC, such as over- or understained slides or slides with dirt or scratches, smear too thick or thin, precipitates present, or poor smearing and staining, were discarded. Good-quality malaria-positive slides were subjected to parasite counting.⁴ The parasite densities were then determined using the following formula: Parasite density = number of parasite counted × 8,000 (parasite/liter of whole blood) / WBC counted.

Slide batches passing through IQC were provided to six different WHO-certified level-1 microscopists for internal validation. The true count value (25% parasitemia count recommended by the WHO) was calculated from a mean number of parasites per microliter. The true counts were assigned to three different groups: <300 parasites/µL were expressed as low counts, 300–999 parasites/µL were expressed as medium counts, and >1,000 parasites/µL were expressed as high counts.⁵ Batches that were validated successfully were added to the inventory. A “batch” is defined as a collection of identical slides prepared from the blood sample collected from a patient.⁵ Labeled and mounted slides with desiccants were placed in slide cabinets for long-term storage.

RESULTS

Between August 2019 and November 2021, 48 batches were prepared (8,196 slides) (one batch contains 200 slides). In 2021, the number of batches prepared was 22 (10 in Jagdalpur, Chhattisgarh, and 12 from the ICMR-NIMR fever clinic). These encompassed nine P. falciparum infections, nine P. vivax infections, one mixed-species infection, and three negative results. In 2020, 18 batches were prepared, mainly from the ICMR-NIMR fever clinic (16 samples) and two from Udalguri, Assam. These consisted of 11 P. vivax infections, one mixed-species infection, and six negatives. In 2019, eight batches from the ICMR-NIMR fever clinic comprised three P. falciparum infections, two P. vivax infections, and three negative results. Overall, P. vivax batches were the most common, at 45.9%, followed by P. falciparum at 25%, malaria-negative batches at 25%, and mixed infections at 4.1% (two P. falciparum + P. vivax) (Table 2).

Table 2.

RDT, microscopy, and PCR results of all slides, including slide inventory details by year

Sample Number	Year	RDT Result	Microscopy Result	PCR Result	Number of Slides in Inventory
1	2019	Negative	Negative	DBS lost	44
2	2019	Negative	Negative	DBS lost	48
3	2019	Negative	Negative	DBS lost	76
4	2019	P. vivax	P. vivax	P. vivax	88
5	2019	P. falciparum	P. falciparum	DBS lost	143
6	2019	P. falciparum	P. falciparum	P. falciparum	159
7	2019	P. vivax	P. vivax	DBS lost	179
8	2019	P. falciparum	P. falciparum	P. falciparum	158
9	2020	P. vivax	P. vivax	P. vivax	152
10	2020	P. vivax	P. vivax	P. vivax	156
11	2020	P. vivax	P. vivax	P. vivax	171
12	2020	P. vivax	P. vivax	P. vivax + P. ovale	165
13	2020	P. vivax	P. vivax	P. vivax	139
14	2020	P. vivax	P. vivax	P. vivax	173
15	2020	P. vivax	P. vivax	P. vivax	188
16	2020	Negative	Negative	Negative	191
17	2020	P. vivax	P. vivax	P. vivax	185
18	2020	P. vivax	P. vivax	P. vivax	182
19	2020	Negative	Negative	Negative	198
20	2020	Negative	Negative	Negative	197
21	2020	Negative	Negative	Negative	198
22	2020	Negative	Negative	Negative	176
23	2020	P. vivax	P. vivax	P. vivax	192
24	2020	P. vivax	P. vivax	P. vivax	190
25	2020	Negative	Negative	Negative	184
26	2020	P. falciparum	P. falciparum + P. vivax	P. falciparum + P. vivax	194
27	2021	P. falciparum	P. falciparum	DBS lost	188
28	2021	P. falciparum	P. falciparum	DBS lost	185
29	2021	P. falciparum	P. falciparum	DBS lost	156
30	2021	P. falciparum	P. falciparum	P. falciparum	186
31	2021	P. falciparum	P. falciparum	DBS lost	190
32	2021	P. falciparum	P. falciparum	P. falciparum	191
33	2021	P. falciparum	P. falciparum	P. falciparum	196
34	2021	P. falciparum	P. falciparum	P. falciparum	195
35	2021	P. falciparum	P. falciparum + P. vivax	P. falciparum + P. vivax	194
36	2021	P. falciparum	P. falciparum	P. falciparum	184
37	2021	P. vivax	P. vivax	P. vivax	200
38	2021	P. vivax	P. vivax	P. vivax	196
39	2021	P. vivax	P. vivax	P. vivax	194
40	2021	P. vivax	P. vivax	P. vivax	200
41	2021	P. vivax	P. vivax	P. vivax	84
42	2021	P. vivax	P. vivax	P. vivax	185
43	2021	P. vivax	P. vivax	P. vivax	177
44	2021	P. vivax	P. vivax	P. vivax	188
45	2021	P. vivax	P. vivax	P. vivax	181
46	2021	Negative	Negative	Negative	200
47	2021	Negative	Negative	Negative	200
48	2021	Negative	Negative	Negative	200

DBS = dried blood sample; P. falciparum = Plasmodium falciparum; P. vivax = Plasmodium vivax; PCR = polymerase chain reaction; RDT = rapid diagnostic test.

All 48 batches passed internal validation, and there was no discrepancy in the results among the six different WHO-certified level-1 microscopists (Figure 1). A total of 12 samples were negative. The true count, the median of the validators’ counts, ranged from 111 to 280,795 parasites/µL. The results based on the three categories of true counts were as follows⁵; 11.4% (4/35) low counts, 14.2% (5/35) medium counts, and 74.2% (26/35) high counts.

Figure 1.

Box plot comparing parasite densities determined by six different microscopists by parasite species.

Of 48 microscopically examined slides, only 39 DBS samples were confirmed by PCR (nine DBS samples were lost) (Table 2). Polymerase chain reaction revealed that 7 samples out of 39 were P. falciparum infections and 20 samples out of 39 were P. vivax infections. Further, mixed infections with P. falciparum and P. vivax were found in two samples (2/39), and those with P. vivax and P. ovale were found in one sample (1/39); 9 samples out of 39 were negative. The PCR results of 38 samples concurred with the validated microscopy results except for one mixed-infection result (P. vivax + P. ovale) (Table 2).

DISCUSSION

The ICMR-NIMR, New Delhi, initiated the development of a malaria slide bank to strengthen QA and malaria diagnosis efforts. India needs to implement a comprehensive large-scale QA system to recruit proficient malaria microscopists and gather quality malaria-positive slides for training microscopists when malaria cases are decreasing significantly. However, it is very difficult for most countries to develop their malaria slide banks. Some of the major limitations to malaria slide banking are limited access to positive samples, lack of infrastructure, funds, dedicated staff, and quality of smear, required blood volume, especially in children, and delay in transferring positive blood samples from the collection site to the slide preparation site.

All batches of slides were fixed at the field sites by using methanol and transported to ICMR-NIMR for staining and further processing. For this reason, fresh methanol is recommended during the fixing of blood films to avoid the loss of their fixative efficacy.⁵ For staining, 3% Giemsa stain was used for 45 minutes, which gave 100% sensitivity and specificity for detecting malaria species. Horning et al. used an automated system (algorithm based) to screen the Giemsa-stained blood films that achieved a 94.3% detection accuracy.⁷ Integration of the malaria slide bank with artificial intelligence-based approaches would strengthen malaria microscopy.⁸

Another major limitation was finding rare species of malaria (P. malariae, P. ovale); a field trip to an area with a high malaria load is strongly advised by the WHO.⁹ The exchange of slides with other established slide banks from other countries is an alternative. Sharing slides of less common species like P. malariae and P. ovale, along with prevalent species in each country, would enhance the slide banks of both nations.

The India’s MSB may also support other Southeast Asian countries bordering India, such as Bhutan, Myanmar, and Bangladesh. The malaria slide bank is an important asset for any country where malaria is endemic; for example, while India is moving towards malaria elimination, low-density Plasmodium infections may lead to misdiagnosis and microscopists may misinterpret mixed-species infection. A study reported that microscopically confirmed P. falciparum species missed 18% of mixed-species infections.¹⁰

Polymerase chain reaction is helpful in the accurate detection of all Plasmodium species, especially for mixed-species infections.¹¹ Mixed infections are typically overlooked, as barely 2% of mixed infections can be detected by microscopy.¹² In India, studies comparing various malaria diagnostic methods showed the effectiveness of PCR in contrast to microscopy¹³ and recommended the use of molecular tests in field settings to prevent misdiagnosis.¹⁰ PCR proved helpful in the detection of non-falciparum species, particularly relapsing malaria species that can overcome microscopy challenges (especially low-density infections and mixed infections).¹⁴ However, clinical research investigations using microscopy as a reference standard form the basis of a quality management system.¹⁵

Numerous requests for high-quality, different parasite count slides have been made by health authorities and regional health offices in Indian states where malaria is endemic. These slides support microscopy training, assessment, and capacity building for microscopists, including the NCAMM, refresher training, and WHO level-1/level-2 technician competency assessments. The success of these services relies on the growth and sustainability of the ICMR-NIMR’s malaria slide bank.

CONCLUSION

The MSB plays a vital role in India’s malaria elimination goal. It supports programs and microscopists by providing a wide range of quality control slides, facilitating training and practical experience. Serving as the primary repository for slides, it is indispensable for national training.